JP4625019B2 - Reagents and methods for Neisseria gonorrhoeae detection - Google Patents

Description

  The present invention relates to the fields of molecular biology and nucleic acid science. The present invention provides methods and reagents for detecting pathogens such as Neisseria gonorrhoeae and also relates to the fields of medical diagnosis and prognosis.

  Neisseria is a human pathogen that is the causative microorganism of gonorrhea, N. cerevisiae. Consists of gram-negative aerobic bacteria, including N. gonorrhoeae. N. has a high prevalence and low mortality. Gonorrhoeae infection is usually caused by sexual contact, typically affecting the urethral mucosa in men and the cervix in women. However, infection can spread to other tissues. For example, genital infections in men can lead to symptoms of prostatitis going up the urethra, while N. Gonorrhoeae infections can spread to the fallopian tube and, if left untreated, can ultimately cause infertility. N. The pathogenic mechanism of Gonoloaeae involves the attachment of bacteria to non-ciliated epithelial cells via the cilia. This mechanism also includes the production of endotoxins and IgA proteases.

  N. Co-infection of Gonoloae and Chlamydia trachomatis is often observed. Both infections are two known causes of ectopic pregnancy and can lead to infertility if untreated. They are also known to be responsible for the acute clinical symptoms of mucinous cervicitis and pelvic inflammatory disease. Therefore, N. can be asymptomatic. Gonoloae and C.I. Detection of C. trachomatis infection is important for individuals in need of treatment, especially in women, and for a wider population at risk of infection occurring and even transmission of the infection.

  Detection and identification of bacterial infections has traditionally been accomplished by pure culture isolation and measurement techniques, which include specimen sources, growth requirements, visible growth characteristics, microscopic morphology, staining Use knowledge of reactions and biochemical characteristics. For example, N.I. Existing methods for detecting and identifying Gonorrhoeae infections include Gram staining, culture on selective agar media, and cytochrome oxidase and carbohydrate utilization tests. Serological assays involving coaggregation and fluorescent antibody staining are also described in N. We have described the detection of Gonoloae. Culture-based methods are relatively sensitive, but are usually time consuming to perform, often involve overnight incubation, and are labor intensive. Gram stain and antibody-based tests typically give results in less than an hour, but are usually less sensitive than culture-based methods.

  The use of specific polynucleotide sequences as probes for the recognition of infectious agents is one alternative to problematic immunological identification assays and other existing methodologies. For example, nucleic acid probes complementary to target nucleic acid sequences have been used in hybridization methods such as Southern blots and dot plots to detect target nucleic acid sequences. Many of these hybridization methods rely on the culture and / or enrichment of organisms and are therefore not suitable for rapid diagnosis. Among many others, the advent of techniques for rapid amplification of specific nucleic acid sequences, such as the polymerase chain reaction, use sequence-specific probes directly on clinical specimens, thereby allowing hybridization assays Provided a mechanism to eliminate pathogen enrichment and in vitro culturing prior to conducting Thus, amplification-based hybridization assays can provide a simple and rapid diagnostic technique for the detection of pathogens in clinical samples.

  Many probes used to date include N.I. Gonoeroe, N.H. It lacks sufficient specificity to distinguish pathogenic agents having highly homologous nucleic acid sequences, such as N. meningitidis, and the like. This can lead to biased assay results, including false positives. One result of such misdiagnosis would be the application of an inappropriate treatment process to the patient.

  The present invention relates to species-specific methods and reagents for rapid detection of Neisseria gonorrhoeae, ie, methods and reagents that do not substantially detect other species of the genus Neisseria, or species from other genera. I will provide a. For example, the nucleic acid detection reagents of the present invention (eg, probe nucleic acids, sequence-specific antibodies, etc.) are typically It binds to a nucleic acid sequence that is present in Gonorrhoeae but not in other species. Furthermore, N.I. Since patients infected with Gonoloae are often also infected with Chlamydia trachomatis, the present invention also provides for N. cerevisiae in the sample. Gonoloae and C.I. A method for simultaneously detecting trachomatis is also provided. This approach minimizes the number of diagnostic methods that a patient can receive and typically also minimizes the overall diagnostic cost. In addition to compositions and reaction mixtures, the present invention also relates to kits and systems for the detection of these pathogenic agents, and relates to computers and computer-readable media.

  In one aspect, the present invention provides an oligonucleotide comprising a nucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 3 to 27 or a complement thereof. In another aspect, the present invention provides an oligonucleotide comprising a nucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 3-27 and its complement, and having no more than 100 nucleotides . In yet another aspect, the invention relates to an oligonucleotide having at least 90% (eg, at least 95%, etc.) sequence identity to one of SEQ ID NOs: 3-27, or its complement. An oligonucleotide having 100 or fewer nucleotides is provided. In such embodiments, typically these oligonucleotides are primer nucleic acids, probe nucleic acids, or the like. In some of these embodiments, the oligonucleotide has 40 or fewer nucleotides (eg, 35 or fewer nucleotides, or 30 or fewer nucleotides, etc.). In some embodiments, the oligonucleotide comprises at least one modified nucleotide. Optionally, the oligonucleotide comprises at least one label and / or at least one quencher component. In some embodiments, the oligonucleotide comprises at least one conservatively modified variation.

  In another aspect, the present invention provides an oligonucleotide comprising at least 90% sequence identity with the following SEQ ID NO: 1, SEQ ID NO: 2, or its complement, and having no more than 100 nucleotides Relates to nucleotides. In some embodiments, the oligonucleotide has a sequence between about 12 and about 50 nucleotides in length. In some embodiments, at least one nucleotide of the oligonucleotide is modified to alter the hybridization stability of the nucleic acid relative to the unmodified nucleotide. In some embodiments, the oligonucleotide comprises at least one label and / or at least a quencher component. In some embodiments, the solid support comprises an oligonucleotide.

  In another aspect, the present invention relates to a method for detecting Neisseria gonorrhoeae in a sample, wherein (a) in at least one nucleic acid amplification reaction, the nucleic acid derived from the sample is represented by SEQ ID NO: 1, SEQ ID NO: 2, substantially Or a variant thereof having at least 90% sequence identity with one of SEQ ID NO: 1 or 2, or a complement of said variant Contacting with at least one first pair of primer nucleic acids that selectively binds to a nucleic acid having a sequence consisting of: The method also includes detecting (b) the nucleic acid and / or one or more amplicons thereof from the nucleic acid amplification reaction during or after (a), thereby detecting Neisseria gonorrhoeae in the sample. . In some embodiments, for example, the nucleic acid and / or its amplicon comprises at least one sequence selected from the group consisting of SEQ ID NOs: 28-33. In some embodiments, (a) comprises contacting the nucleic acid from the sample with a second pair of primer nucleic acids that are at least partially complementary to Chlamydia trachomatis. In these embodiments, (b) comprises detecting one or more additional amplicons from the nucleic acid amplification reaction during or after (a), thereby detecting Chlamydia trachomatis in the sample. . In some embodiments, at least one primer nucleic acid comprises a modified primer nucleic acid. In some embodiments, the at least one primer nucleic acid comprises at least one label. In these embodiments, (b) optionally detects a detectable signal generated by the label, or amplifies the detectable signal generated by the label to generate an amplified signal, and detects the amplified signal. Comprising doing. In some embodiments, (b) comprises at least 90% sequence identity with an amplicon and SEQ ID NO: 1, SEQ ID NO: 2, a substantially identical variant thereof, and one of SEQ ID NO: 1 or 2. A binding between one or a plurality of nucleic acid detection reagents that detectably bind to a nucleic acid having a sequence comprising SEQ ID NO: 1, SEQ ID NO: 2, or a complement of the mutant. Monitoring. Typically, the at least one nucleic acid detection reagent comprises at least one label and / or at least one quencher component. In these embodiments, (b) optionally detects a detectable signal generated by the label, or amplifies the detectable signal generated by the label to generate an amplified signal, and detects the amplified signal. Comprising doing.

  In another aspect, the present invention provides a method for determining the presence of Neisseria gonorrhoeae in a sample comprising: (a) a sample-derived nucleic acid and / or an amplicon thereof, SEQ ID NO: 1, SEQ ID NO: 2, A variant thereof that is at least 90% identical to one of SEQ ID NO: 1 or 2, or a complement of SEQ ID NO: 1, SEQ ID NO: 2, or said variant There is provided a method comprising contacting with one or more oligonucleotides that selectively bind to a nucleic acid having a bovine sequence. The method also includes (b) binding between the nucleic acid and / or its amplicon and the oligonucleotide (eg, at one time point, at a number of separate time points, continuously over a selected time, etc.) ) Where the detectable binding between the nucleic acid and / or its amplicon and the oligonucleotide determines the presence of Neisseria gonorrhoeae in the sample. In some embodiments, for example, the nucleic acid and / or its amplicon comprises at least one sequence selected from the group consisting of SEQ ID NOs: 28-33. The presence of Neisseria gonorrhoeae in the sample is usually unknown or unproven before (a). In some embodiments, (a) comprises contacting the nucleic acid and / or its amplicon with an oligonucleotide in solution at a temperature of at least 42 ° C. for at least 15 minutes, wherein The weight comprises about 50% formalin and heparin at a concentration of about 1 mg / ml. In addition, the method typically comprises a reaction other than a sequencing reaction. The sample is usually derived from a mammalian subject, such as a human subject. In some embodiments, the nucleic acid and / or its amplicon and the oligonucleotide are contacted in solution. Optionally, the solid support comprises nucleic acids and / or amplicons (eg, arrayed on the solid support). As an additional option, the solid support comprises an oligonucleotide.

  In certain embodiments of the invention, the method further comprises contacting the sample-derived nucleic acid and / or its amplicon with at least one additional oligonucleotide that detectably binds to a Chlamydia trachomatis nucleic acid. Become. In these embodiments, the method also includes monitoring binding between the nucleic acid and / or its amplicon and the additional oligonucleotide, thereby detecting Chlamydia trachomatis in the sample. In some embodiments, the method repeats (a) and (b) at least one or more times with at least one additional sample (eg, from the same subject), and the nucleic acid of (b) And / or the binding between the amplicon and the oligonucleotide is diagnosed for eg Neisseria gonorrhoeae and / or Chlamydia trachomatis, or the like, compared to (b) repeated at least once Monitoring the course of treatment of the treated subject.

  The nucleic acid detection reagent of the present invention includes various embodiments. Illustratively, at least one nucleic acid detection reagent comprises an oligonucleotide (eg, probe nucleic acid, primer nucleic acid, etc.). Typically, the oligonucleotide is SEQ ID NO: 1, SEQ ID NO: 2, a variant thereof substantially identical and having at least 90% sequence identity with one of SEQ ID NO: 1 or 2. It has at least 85% (eg, about 90%, about 95%, etc.) sequence identity with a variant, or a subsequence of SEQ ID NO: 1, SEQ ID NO: 2, or the complement of the variant. In some embodiments, (b) comprises monitoring binding between the oligonucleotide and the nucleic acid and / or its amplicon. Optionally, the oligonucleotide has a sequence between about 8 and about 100 nucleotides in length. In certain embodiments, the oligonucleotide is SEQ ID NO: 3-27, a variant thereof substantially identical that has at least 90% sequence identity to one of SEQ ID NOs: 3-27, And a sequence selected from the group consisting of SEQ ID NOs: 3 to 27 and the complement of the mutant. Optionally, at least one oligonucleotide is modified. In some embodiments, for example, the nucleotide is modified to alter hybridization stability compared to the unmodified nucleotide.

  Illustratively, the at least one nucleic acid detection reagent is optionally of SEQ ID NO: 1: 259, 260, 262, 264, 265, 266, 268, 269, 273, 275, 276, 277, 279, 297, 298. 300, 301, 302, 303, 304, 305, 306, 308, 313, 314, 315, 316, 317, 318, 320, 321, 325, 326, 428, 429, 431, 432, 433, 434, 435 Detectably binds to a nucleic acid segment comprising one or more nucleotide positions selected from the group consisting of 440, 441, and 447. As an additional option, the at least one nucleic acid detection reagent is SEQ ID NO: 2: 89, 90, 91, 92, 95, 98, 101, 105, 106, 107, 216, 217, 220, 222, 223, 225. 233, 235, 236, 238, 335, 336, 337, 338, 339, 342, 345, 346, and a nucleic acid segment comprising one or more nucleotide positions selected from the group consisting of 351 To join. In other embodiments, the at least one nucleic acid detection reagent comprises, for example, a sequence specific antibody.

  In some embodiments, the nucleic acid, its amplicon, and / or nucleic acid detection reagent comprises at least one label and / or at least one quencher component. For example, the label may optionally be a fluorescent dye, weak fluorescent label, non-fluorescent label, colorimetric label, chemiluminescent label, bioluminescent label, antibody, antigen, biotin, hapten, mass modifying group, radioisotope, enzyme, or other The same thing is included. In these embodiments, (b) typically comprises detecting a detectable signal generated by the label. Illustratively, (b) optionally comprises (i) amplifying the detectable signal generated by the label to generate an amplified signal, and (ii) detecting the amplified signal.

  In some embodiments, at least one nucleic acid is amplified to produce an amplicon prior to or during (a) using at least one nucleic acid amplification technique, and (b) is the nucleic acid and / or its amplifier Monitoring the binding between the recon and the nucleic acid detection reagent during or after amplification. For example, nucleic acid amplification techniques typically comprise polymerase chain reaction, ligase chain reaction, and / or the like. In these embodiments, the segment is SEQ ID NO: 3-27, a variant thereof substantially identical that has at least 90% sequence identity to one of SEQ ID NOs: 3-27, Further, amplification is optionally performed using at least one primer nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs: 3 to 27 and the complement of the mutant. In some embodiments, the primer nucleic acid comprises at least one label as described herein or known in the art. Optionally, the primer nucleic acid comprises a modified primer nucleic acid (eg, nucleic acid amplification specificity-changing modification, restriction site linker, and / or the like).

  In another aspect, the present invention relates to a method for detecting Neisseria gonorrhoeae in a sample. The method comprises: (a) subjecting a sample-derived nucleic acid to at least 90% sequence identity to one of SEQ ID NOs: 3-27, substantially identical variants thereof, SEQ ID NOs: 3-27; And contacting a first pair of primer nucleic acids comprising at least one nucleic acid selected from the group consisting of SEQ ID NOs: 3 to 27 and a complement of said variant. Furthermore, the method also comprises detecting (b) the nucleic acid and / or one or more amplicons thereof during or after (a) from the nucleic acid amplification reaction, whereby the Neisseria. Detect Goronoaae. In some embodiments, for example, the nucleic acid and / or its amplicon comprises at least one sequence selected from the group consisting of SEQ ID NOs: 28-33. The sample is typically derived from a mammalian subject, such as a human subject. Typically, at least one primer nucleic acid comprises a modified primer nucleic acid. In some embodiments, for example, the modified primer nucleic acid comprises a nucleic acid amplification specificity-changing modification and / or a restriction site linker modification. In some embodiments, (a) contacting a nucleic acid from a sample with at least one second pair of primer nucleic acids that is at least partially complementary to a Chlamydia trachomatis nucleic acid, and (b) Or detecting a plurality of additional amplicons from the nucleic acid amplification reaction during or after (a), thereby detecting Chlamydia trachomatis in the sample.

  In some embodiments, the at least one primer nucleic acid comprises at least one label. Labels optionally include, for example, fluorescent dyes, weak fluorescent labels, non-fluorescent labels, colorimetric labels, chemiluminescent labels, bioluminescent labels, antibodies, antigens, biotin, haptens, mass modifying groups, radioisotopes, enzymes, etc. It becomes. In these embodiments, (b) typically comprises detecting a detectable signal generated by the label. Optionally (b) comprises (i) amplifying the detectable signal generated by the label to generate an amplified signal, and (ii) detecting the amplified signal.

  In some embodiments, (b) is an amplicon and SEQ ID NO: 1, SEQ ID NO: 2, a substantially identical variant thereof, and having at least 90% sequence identity with one of SEQ ID NO: 1 or 2. A binding between one or a plurality of nucleic acid detection reagents that detectably bind to a variant having, or a nucleic acid having a sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, or a complement of the variant. Comprising monitoring. Optionally, the at least one nucleic acid detection reagent comprises an oligonucleotide (eg, a probe nucleic acid, etc.). In some of these embodiments, (b) comprises detecting hybridization between the oligonucleotide and the amplicon. Optionally, the oligonucleotide comprises a sequence between about 8 and about 100 nucleotides in length. In some embodiments, at least one of the oligonucleotides is modified (eg, to alter nucleic acid hybridization stability relative to unmodified nucleotides, or the like). For example, the at least one nucleic acid detection reagent is SEQ ID NO: 3-27, a variant thereof that is substantially identical and having at least 90% sequence identity to one of SEQ ID NOs: 3-27 And a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs: 3 to 27 and the complement of the mutant. Illustratively, the at least one nucleic acid detection reagent is optionally of SEQ ID NO: 1: 259, 260, 262, 264, 265, 266, 268, 269, 273, 275, 276, 277, 279, 297, 298. 300, 301, 302, 303, 304, 305, 306, 308, 313, 314, 315, 316, 317, 318, 320, 321, 325, 326, 428, 429, 431, 432, 433, 434, 435 Detectably binds to a nucleic acid segment comprising one or more nucleotide positions selected from the group consisting of 440, 441, and 447. As an additional option, the at least one nucleic acid detection reagent is SEQ ID NO: 2: 89, 90, 91, 92, 95, 98, 101, 105, 106, 107, 216, 217, 220, 222, 223, 225. 233, 235, 236, 238, 335, 336, 337, 338, 339, 342, 345, 346, and a nucleic acid segment comprising one or more nucleotide positions selected from the group consisting of 351 To join. In some embodiments, the at least one nucleic acid detection reagent comprises a sequence specific antibody or the like. Optionally, the at least one nucleic acid detection reagent comprises at least one label and / or quencher component. Representative labels are optionally fluorescent dyes, weak fluorescent labels, non-fluorescent labels, colorimetric labels, chemiluminescent labels, bioluminescent labels, antibodies, antigens, biotin, haptens, mass modifying groups, radioisotopes, enzymes, or Others include the same. In these embodiments, (b) typically comprises detecting a detectable signal generated by the label. Optionally (b) comprises (i) amplifying the detectable signal generated by the label to generate an amplified signal, and (ii) detecting the amplified signal.

  In another aspect, the present invention is a method for detecting Neisseria gonorrhoeae in a sample, wherein (a) in at least one nucleic acid amplification reaction, the nucleic acid from the sample is converted to at least one first pair of primer nucleic acids. Wherein each primer nucleic acid has a nucleotide length between 12 and 100, and at least one primer nucleic acid is SEQ ID NO: 1, SEQ ID NO: 2, or a complement thereof And at least 90% sequence identity. The method also includes detecting (b) the nucleic acid and / or one or more amplicons thereof from the nucleic acid amplification reaction during or after (a), thereby detecting Neisseria gonorrhoeae in the sample. . Typically, the presence of Neisseria gonorrhoeae in the sample is unknown or unproven prior to (a). In some embodiments, the one or more primer nucleic acids is SEQ ID NO: 3-27, a substantially identical variant thereof having at least 90% sequence identity to one of SEQ ID NOs: 3-27 A variant, and a sequence selected from the group consisting of SEQ ID NOs: 3-27 and the complement of the variant.

  In yet another aspect, the present invention provides a method for detecting the presence of Neisseria gonorrhoeae in a sample, comprising (a) nucleic acid from a sample and / or its amplicon comprising between 12 and 100 nucleotides. And contacting with at least one oligonucleotide having at least 90% sequence identity with a partial sequence of SEQ ID NO: 1, SEQ ID NO: 2, or its complement. Furthermore, the method also includes (b) monitoring the binding between the nucleic acid and / or its amplicon and the oligonucleotide, wherein the nucleic acid and / or its amplicon and the oligonucleotide are between The detectable binding determines the presence of Neisseria gonorrhoeae in the sample. Typically, the presence of Neisseria gonorrhoeae in the sample is unknown or unproven prior to (a). In some embodiments, the one or more primer nucleic acids is SEQ ID NO: 3-27, a substantially identical variant thereof having at least 90% sequence identity to one of SEQ ID NOs: 3-27. And a sequence selected from the group consisting of SEQ ID NOs: 3 to 27 and a complement of the variant.

  In another aspect, the present invention relates to a sample from a subject, SEQ ID NO: 1, SEQ ID NO: 2, a substantially identical variant thereof, and at least 90 of one of SEQ ID NO: 1 or 2 % One or more that binds selectively to a nucleic acid having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, or the complement of the mutant. And a nucleic acid detection reagent. The presence of Neisseria gonorrhoeae in the sample is usually unknown or unproven before (a). Typically, the at least one nucleic acid detection reagent comprises an oligonucleotide (eg, a probe nucleic acid, a primer nucleic acid, or the like). Typically, the oligonucleotide comprises at least 85% (eg, about 90%, about 95%, etc.) sequence identity with a partial sequence of SEQ ID NO: 1, SEQ ID NO: 2, or its complement. In some of these embodiments, the oligonucleotide has a sequence between about 8 and about 100 nucleotides in length (eg, between about 12 and about 50 nucleotides in length). In some embodiments, at least one nucleotide of the oligonucleotide is modified (eg, to alter nucleic acid hybridization stability relative to unmodified nucleotides). For example, the nucleic acid detection reagent is optional: the nucleic acid detection reagent is optional: SEQ ID NO: 3-27, a substantially identical variant thereof and at least 90% sequence relative to one of SEQ ID NO: 3-27 A variant having identity, and at least one nucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 3 to 27 and the complement of the variant. Illustratively, the at least one nucleic acid detection reagent is optionally of SEQ ID NO: 1: 259, 260, 262, 264, 265, 266, 268, 269, 273, 275, 276, 277, 279, 297, 298. 300, 301, 302, 303, 304, 305, 306, 308, 313, 314, 315, 316, 317, 318, 320, 321, 325, 326, 428, 429, 431, 432, 433, 434, 435 Detectably binds to a nucleic acid segment comprising one or more nucleotide positions selected from the group consisting of 440, 441, and 447. As an additional option, the at least one nucleic acid detection reagent is SEQ ID NO: 2: 89, 90, 91, 92, 95, 98, 101, 105, 106, 107, 216, 217, 220, 222, 223, 225. 233, 235, 236, 238, 335, 336, 337, 338, 339, 342, 345, 346, and a nucleic acid segment comprising one or more nucleotide positions selected from the group consisting of 351 To join. In some embodiments, the nucleic acid detection reagent comprises at least one sequence specific antibody. In some embodiments, the composition further comprises at least one additional nucleic acid detection reagent that selectively binds to Chlamydia trachomatis nucleic acid.

  Typically, the at least one nucleic acid detection reagent comprises at least one label and / or quencher component. Illustratively, the label can be any fluorescent dye, weak fluorescent label, non-fluorescent label, colorimetric label, chemiluminescent label, bioluminescent label, antibody, antigen, biotin, hapten, mass modifying group, radioisotope, enzyme Or the like.

  The nucleic acid detection reagent of the composition of the present invention is provided in various ways. In some embodiments, for example, at least one nucleic acid detection reagent is in solution. In other embodiments, the solid support comprises at least one nucleic acid detection reagent. In these embodiments, the nucleic acid detection reagent is noncovalently or covalently attached to the solid support. Exemplary solid supports utilized in these embodiments include, for example, plates, microwell plates, beads, microbeads (eg, magnetic microbeads), tubes (eg, macrotubes, etc.), fibers, whiskers, combs , Arbitrarily selected from hybridization chips, membranes, single crystals, ceramic layers, self-assembled monolayers, and the like.

  Illustratively, the nucleic acid detection reagent is optionally conjugated with biotin or a biotin derivative, and the solid support is optionally conjugated with avidin or avidin derivative, or streptavidin or streptavidin derivative. In some embodiments, the linker attaches the nucleic acid detection reagent to the solid support. The linker is typically selected from oligopeptides, oligonucleotides, oligopolyamides, oligoethyleneglycerols, oligoacramides, alkyl chains, and the like. Optionally, a cleavable attachment attaches the nucleic acid detection reagent to the support. A cleavable attachment is typically cleaved by, for example, heat, enzymes, chemicals, or electromagnetic radiation.

  In another aspect, the invention provides SEQ ID NO: 1, SEQ ID NO: 2, a substantially identical variant thereof having at least 90% sequence identity with one of SEQ ID NO: 1 or 2. Or a set of amplicons having a sequence corresponding to a subsequence of SEQ ID NO: 1, SEQ ID NO: 2, or the complement of said variant. Typically, at least one subset of the set of amplicons is SEQ ID NO: 3-27, a substantially identical variant thereof and at least 90% sequence relative to one of SEQ ID NO: 3-27 It is produced using a mutant having identity, and at least one primer nucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 3 to 27 and the complement of the mutant. In some embodiments, the primer nucleic acid comprises a modified primer nucleic acid. For example, a modified primer nucleic acid optionally comprises a nucleic acid amplification specificity alteration modification, a restriction site linker modification, and / or the like. In some embodiments, the reaction mixture further comprises an additional set of amplicons comprising sequences corresponding to Chlamydia trachomatis nucleic acid sequences.

  In another aspect, the invention is a kit comprising (a) between 12 and 100 or fewer nucleotides, and at least 90% of a partial sequence of SEQ ID NO: 1, SEQ ID NO: 2, or its complement At least one oligonucleotide comprising the sequence identity of: and (b) one or more of: (b) by monitoring the binding between the nucleic acid from the sample and / or its amplicon and said oligonucleotide; Instructions for determining the presence of Neisseria gonorrhoeae in a sample, whether present or not, or (c) a kit comprising at least one container for packing at least one oligonucleotide . In some of these embodiments, the oligonucleotide has a sequence between about 8 and 100 nucleotides in length. In some embodiments, for example, the oligonucleotide is SEQ ID NO: 3-27, a variant thereof substantially identical that has at least 90% sequence identity to one of SEQ ID NOs: 3-27 And a sequence selected from the group consisting of SEQ ID NOs: 3-27 and the complement of the mutant. Illustratively, the oligonucleotide is optionally of SEQ ID NO: 1: 259, 260, 262, 264, 265, 266, 268, 269, 273, 275, 276, 277, 279, 297, 298, 300, 301. , 302, 303, 304, 305, 306, 308, 313, 314, 315, 316, 317, 318, 320, 321, 325, 326, 428, 429, 431, 432, 433, 434, 435, 440, 441 , And 447 to a nucleic acid segment comprising one or more nucleotide positions selected from the group consisting of 447. As an additional option, the oligonucleotide may be of SEQ ID NO: 2: 89, 90, 91, 92, 95, 98, 101, 105, 106, 107, 216, 217, 220, 222, 223, 225, 233, 235. 236, 238, 335, 336, 337, 338, 339, 342, 345, 346, and 351, detectably bind to a nucleic acid segment comprising one or more nucleotide positions selected from the group consisting of. In another embodiment, the nucleic acid detection reagent is a sequence specific antibody. In some embodiments, the kit further comprises one or more nucleic acid detection reagents that specifically bind to Chlamydia trachomatis nucleic acid. In these embodiments, the kit typically further detects Chlamydia trachomatis in the sample by monitoring binding between the sample-derived nucleic acid and / or its amplicon and an additional nucleic acid detection reagent. Instructions and / or one or more containers for packing additional nucleic acid detection reagents. In some embodiments, the kit typically further comprises at least one enzyme (eg, a polymerase, etc.) and / or one or more nucleotides.

  In some embodiments, the nucleic acid detection reagent is in solution, whereas on the other hand the solid support comprises the nucleic acid detection reagent. The solid support is optionally, for example, a plate, microwell plate, bead, microbead, tube, fiber, whisker, comb, hybridization chip, membrane, single crystal, ceramic layer, self-assembled monolayer, or the like Arbitrarily selected from

  Typically, the oligonucleotide comprises at least one label and / or quencher component. Exemplary labels are, for example, fluorescent dyes, weak fluorescent labels, non-fluorescent labels, colorimetric labels, chemiluminescent labels, bioluminescent labels, antibodies, antigens, biotin, haptens, mass modifying groups, radioisotopes, enzymes, or Others include the same.

  In yet another aspect, the present invention provides a system (eg, an automated system) for detecting Neisseria gonorrhoeae in a sample. The system comprises (a) at least one oligonucleotide having between 12 and 100 or fewer nucleotides, and at least 90% sequence identity with the sequence of SEQ ID NO: 1, SEQ ID NO: 2, or its complement Oligonucleotides comprising sex. The system also includes (b) at least one detector that detects binding between the nucleic acid from the sample and / or its amplicon and the oligonucleotide, and (c) at least operably connected to the detector. Also included is a controller that includes one or more instruction sets that correlate the binding detected by the detector with the presence of Neisseria gonorrhoeae in the sample. The oligonucleotide typically has a sequence selected from the group consisting of SEQ ID NOs: 3-27, or complements thereof. To further illustrate, the oligonucleotide is typically of SEQ ID NO: 1: 259, 260, 262, 264, 265, 266, 268, 269, 273, 275, 276, 277, 279, 297, 298, 300. 301, 302, 303, 304, 305, 306, 308, 313, 314, 315, 316, 317, 318, 320, 321, 325, 326, 428, 429, 431, 432, 433, 434, 435, 440 , 441, and 447, detectably binds to a nucleic acid segment comprising one or more nucleotide positions selected from the group consisting of. As an additional option, the oligonucleotide may be of SEQ ID NO: 2: 89, 90, 91, 92, 95, 98, 101, 105, 106, 107, 216, 217, 220, 222, 223, 225, 233, 235. 236, 238, 335, 336, 337, 338, 339, 342, 345, 346, and 351, detectably bind to a nucleic acid segment comprising one or more nucleotide positions selected from the group consisting of. In addition, the oligonucleotide typically comprises at least one label and / or at least one quencher component. In certain embodiments, the system further comprises one or more additional nucleic acid detection reagents that specifically bind to Chlamydia trachomatis nucleic acid, wherein the detector comprises nucleic acid from a sample and / or Detecting a binding between the amplicon and an additional nucleic acid detection reagent, and the controller correlates the binding detected by the detector with the presence of Chlamydia trachomatis in the sample. Comprising one instruction set. In some embodiments, at least one container or solid support comprises an oligonucleotide. In these embodiments, the system optionally (d) at least one temperature regulator operably connected to the container or solid support to adjust the temperature in or on the container or the solid support, and / or Or (e) transporting liquid to and / or from the container or support, eg, to perform one or more nucleic acid amplification techniques in or on the container. At least one liquid transport component.

  In another aspect, the present invention provides (a) SEQ ID NO: 1, SEQ ID NO: 2, a substantially identical variant thereof, having at least 90% sequence identity with one of SEQ ID NO: 1 or 2. A data set comprising a plurality of character strings corresponding to a plurality of sequences corresponding to a partial sequence of SEQ ID NO: 1, SEQ ID NO: 2, or a complement of the mutant A system comprising a computer or computer readable medium is provided. The system also includes (b) an automatic synthesizer connected to a computer or computer readable medium, corresponding to one or more character strings in the data set from the computer or computer readable medium. An automatic synthesizer that receives instructions for instructing the synthesis of a plurality of nucleic acids. Typically, at least one character string corresponds to a sequence selected from the group consisting of: SEQ ID NO: 3-27, or a complement thereof.

I. Definitions Before describing the present invention in detail, the present invention is not limited to a particular oligonucleotide probe, method, composition, reaction mixture, kit, system, computer, or computer-readable medium, and can of course be modified. It should be understood. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments and is not intended to be limiting. Moreover, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In describing and claiming the present invention, the following terminology and grammatical variations will be used in accordance with the definitions set out below.

  A “5′-nuclease probe” has at least two labels and has increased radiation after one of the two labels is cleaved or otherwise separated from the probe. Refers to an oligonucleotide probe that releases In some embodiments, for example, the 5'-nuclease probe is labeled with two different fluorescent dyes, such as a 5 'terminal reporter dye and a 3' terminal quenching dye or component. When the probe is intact, energy transfer typically occurs between the two fluorophores, causing the fluorescence emission from the reporter dye to be quenched. For example, during the extension phase of the polymerase chain reaction, the 5'-nuclease probe bound to the template nucleic acid is cleaved, for example by the 5 'nuclease activity of Taq polymerase, so that fluorescence emission from the reporter dye is no longer quenched. Representative 5′-nuclease probes are, for example, “HOMOGENEOUS ASSAY SYSTEM USING THE NUCLEASE ACTIVITY OF A NUCLEIC” issued May 11, 1993 to Gelfand et al. US Patent No. 5,210,015 entitled "ACID POLYMERASE", "HOMOGENEOUS METHODS FOR NUCLEIC ACID" issued on November 30, 1999 to Higuchi US Pat. No. 5,994,056 entitled “AMPLIFICATION AND DETECTION” and “METHODS AND DEVICES FOR HEMOGENEOUS” issued on 9 January 2001 to Higuchi “NUCLEIC ACID AMPLIFICATION AND DETECTOR” It is described in that U.S. Patent No. 6,171,785.

  The term “change” refers to a change in a nucleic acid sequence, including but not limited to substitutions, insertions, and / or deletions.

  “Amplification reaction” refers to a primer-initiated replication of one or more labeled nucleic acid sequences or their complements.

  An “amplicon” can occur, for example, in transcription, cloning, and / or polymerase chain reaction (“PCR”) (eg, strand displacement PCR amplification (SDA), double stranded PCR amplification, etc.) or other nucleic acid amplification techniques. It refers to a molecule created by duplicating or transcribing another molecule. Typically, an amplicon is a copy of a selected nucleic acid (eg, a template or labeled nucleic acid) or is a complement thereto.

  "Amplification signal" refers to an increased detectable signal that can be generated without or in combination with an amplification reaction. Representative signal amplification techniques include, for example, "Clinical evaluation of branched DNA signal amplification for quantifying HIV type 1 in human plasma" Clinical evaluation of branched DNA signal amplification for quantifying) ", AIDS Res Hum Retroviruses, 1995, Vol. 11, No. 3, p. 353-361 and US Pat. No. 5,437,977 to Segev, US Pat. No. 6,033,853 to Delair et al., And US Pat. No. 6 to Horn. 180,777.

  An “antibody” is a polypeptide substantially encoded by at least one immunoglobulin gene, or a fragment of at least one immunoglobulin gene, that is capable of participating in detectable binding to a ligand. Point to. The term includes naturally occurring forms, as well as fragments and derivatives. Fragments are within the terms used herein, such as those produced by digestion with peptidases, such as Fab, Fab ′, and F (ab) ′ 2 fragments, such as antigens that indicate disease. As long as detectable binding to the target molecule remains possible, including those produced by chemical separation, chemical cleavage.

  “Array” refers to a population of elements. The population can be spatially aligned (“patterned array”) or irregular (“randomly patterned” array). The array can form or comprise one or more functional elements (eg, probe regions on the microarray).

  The term “attached” or “joined” refers to an interaction and / or state in which substances or compounds are linked together or otherwise joined. These interactions and / or states are typically produced by, for example, covalent bonds, ionic bonds, chemisorption, physisorption, and combinations thereof. In some embodiments, for example, the oligonucleotide probe is attached to a solid support. In some embodiments, the oligonucleotide probe is conjugated to biotin (ie, biotinylated), the solid support is conjugated to avidin, and the probe is attached to the solid support, for example, through a binding interaction between biotin and avidin. To do.

  Molecular species “bind” when they associate with each other through covalent and / or non-covalent interactions. For example, two complementary single stranded nucleic acids hybridize to each other to form one nucleic acid having at least one double stranded region. To further illustrate, antibodies and corresponding antigens can also associate non-covalently with each other.

  The term “cleavage” refers to the process of releasing a substance or compound from attachment to another substance or compound. In certain embodiments, for example, the oligonucleotide is cleaved, eg, from a solid support, allowing analysis of the oligonucleotide by a liquid phase method. For example, Wells et al., “Cleavage and Analysis of Material from Single Resin Beads,” incorporated by reference, J. Org. Chem. 1998. 63, p. See 6430.

  “Character”, when used in connection with a character of a string, refers to a subunit of the string. In one embodiment, the characters of the string encode one subunit of the encoded biomolecule. Thus, for example, if the encoded biomolecule is a polynucleotide or oligonucleotide, one letter in the column encodes a single nucleotide.

  A “character string” is any entity capable of storing sequence information (eg, a subunit structure of a biomolecule, such as a nucleotide sequence such as a nucleic acid). In one embodiment, the string can be a simple array of letters (letters, numbers, or other symbols), or such information in a tangible or intangible form (eg, electronic, Can be a numerical or coded representation of a magnetic, etc.). Strings do not necessarily have to be “linear”, but in many other shapes, such as linked lists or other non-linear arrays (eg, used as code to generate a linear array of characters) It can also exist. A string typically encodes an oligonucleotide or polynucleotide string, either directly or indirectly, and is converted to any encrypted string or image or string rather than obscuring. Contains sequences of objects (objects, objects) that can be stored in a polynucleotide, or the like (whether made of natural or artificial monomers) Represents a sequence of monomers or polymers.

The term “Chlamydia trachomatis”, “C. trachomatis”, or “CT” refers to the bacterial species Trachomatis of the genus Chlamydia. See, for example, Stephens et al., "Genome sequence of an obligate intracellular pathogen of humans: Clamydia trachomatis", Science 1998, 282, p. 754-759, Kalmam et al., “Comparative genomes of Chlamydia pneumoniae and C. trachomatis”, Nature Genetics, 1999, Vol. 21, p. 385-389, and Stephens, “Chlamydia: Intracellular Biology, Pathogenesis, and Immunity”, ASM Press, 1999. Typical GenBank (R) accession number for the complete sequence of the genome of Chlamydia trachomatis is a NC _ 000117. In addition, stdgen. lanl. See also the Chlamydia Trachomatis database on gov on the World Wide Web.

  The term “Chlamydia trachomatis nucleic acid” or “C. trachomatis nucleic acid” refers to a nucleic acid (and / or its amplicon) derived from or isolated from Chlamydia trachomatis.

  The term “complement thereof” refers to a nucleic acid that is the same length as a given nucleic acid and is exactly complementary to the given nucleic acid.

  “Composition” refers to a combination of two or more different ingredients. In certain embodiments, for example, the composition comprises a solid support, which comprises, for example, one or more oligonucleotide probes covalently or non-covalently attached to the surface of the support. In other embodiments, the composition comprises one or more oligonucleotides in solution.

  The term “deletion” in the context of a nucleic acid sequence means that at least one nucleotide is from the nucleic acid sequence, eg from the 5′-end of the nucleic acid sequence, from the 3′-end, and / or within the nucleic acid sequence. From position, it refers to the change that is removed.

  The term “derivative” can be produced from a chemical substance that is structurally related to another substance or from another substance (ie, the substance from which it is derived), eg, by chemical or enzymatic modification. Refers to possible chemical substances. Illustratively, the oligonucleotide probe is optionally conjugated with biotin or a biotin derivative. To further illustrate, one nucleic acid is “derived from” another by a process such as chemical synthesis based on knowledge of the sequence of another nucleic acid, amplification of another nucleic acid, or the like. Is possible.

  The term “detectably binds” refers to at least two molecular species (eg, a probe nucleic acid and a target nucleic acid) that can be detected over a background signal (eg, noise) using one or more detection methods. , Binding between sequence-specific antibodies and target nucleic acids, etc.).

  A nucleic acid is “elongated” or “extended” when additional nucleotides (or other similar molecules) are incorporated into the nucleic acid. For example, a nucleic acid is optionally extended by a biocatalyst that incorporates a nucleotide, such as a polymerase that typically adds a nucleotide at the 3 'end of the nucleic acid.

  An “extended primer nucleic acid” refers to a primer nucleic acid to which one or more additional nucleotides have been added or otherwise incorporated (eg, covalently linked thereto).

  Nucleic acids “hybridize” or “bind” when they associate with each other, typically in solution. Nucleic acids hybridize due to a variety of well-characterized physicochemical forces such as hydrogen bonding, solvent elimination, base stacking, and the like. Extensive guidance for nucleic acid hybridization is included in the reference by Tijssen, "Laboratory Techniques in Biochemistry and Molecular Biology --- Hybridization with Nucleic Acid Probes (laboratory in biochemistry and molecular biology). Technology—Hybridization with Nucleic Acid Probes ”, Part 1, Chapter 2,“ Overview of principles of hybridization and the strategy of nucleic acid probe assays ”, Elsevier , New York, 1993, and Ausubel (eds), “Current Protocols in Molecular Biology”, Volumes I, II, and III, 1997. Hames & Higgins, "Gene Probes 1", Oxford University Press IRL Press, Oxford, UK, 1995 (Hames and Higgins 1), and Hames & Higgins "Gene Probes 2", Oxford University Press IRL Press, Oxford, UK, 1995 (Hames and Higgins 2), DNA and RNA synthesis, including oligonucleotides, labeling, detection, and Details are presented for quantification. Both Bums and Higgins, 1 and 2, are included as references.

  “Stringent hybridization wash conditions” are sequence-dependent in the context of nucleic acid hybridization assays or experiments, such as nucleic acid amplification reactions, Southern and Northern hybridizations, or the like, and differ in environmental parameters. Below is different. Extensive guidance for nucleic acid hybridization is found in Tijssen (1993), supra, and in Bums and Higgins, 1 and 2.

For purposes of the present invention, typically “highly stringent” hybridization and wash conditions are at least about 5 ° C. lower than the thermal melting point (T _m ) for a particular sequence at a defined ionic strength and pH. Selected to be. T _m is the temperature (at a defined ionic strength and pH) at which 50% of the test sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the T _m for a particular probe.

  An example of stringent hybridization conditions for hybridization of complementary nucleic acids on filters in Southern or Northern blots is 50% formalin with 1 mg heparin at 42 ° C. and hybridization is performed overnight. It is. An example of stringent wash conditions is a 0.2 × SSC wash at 65 ° C. for 15 minutes (Sambrook et al., “Molecular Cloning: A Laboratory, included as a reference for SSC buffer descriptions. Manual (Molecular Cloning; Laboratory Manual), 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example of a low stringency wash is 2 × SSC at 40 ° C. for 15 minutes. Typically, a signal-to-noise ratio of 5x (or higher) than that observed for an irrelevant probe in a particular hybridization assay indicates the detection of specific hybridization.

  Comparative hybridization can be used to identify the nucleic acids of the invention.

  In particular, the detection of stringent hybridization in the context of the present invention shows strong structural similarity, for example, to the nucleic acids given in the sequences listed herein. For example, nucleic acids that hybridize to the representative nucleic acids herein are preferably identified under stringent conditions. One indication of stringent hybridization is that it can be detected under stringent conditions for one of the listed nucleic acids (eg, a nucleic acid having a sequence selected from SEQ ID NOs: 3-27 and its complement). The ability to hybridize. Stringent hybridization and wash conditions can be readily determined empirically for any test nucleic acid.

  For example, in determining highly stringent hybridization and wash conditions, the stringency of hybridization and wash conditions is gradually increased (eg, increasing temperature) until a combination of criteria selected is encountered. Reducing the salt concentration, increasing the detergent concentration, and / or increasing the concentration of organic solvents such as formalin in hybridization or washing). For example, the stringency of the hybridization and washing conditions is such that a probe consisting of or comprising one or more nucleic acid sequences selected from SEQ ID NOs: 3-27 and its complementary polynucleotide sequences is perfectly matched Hybridization of the probe to a non-target nucleic acid against a complementary target (again, a nucleic acid comprising one or more nucleic acid sequences selected from SEQ ID NOs: 3-27 and its complementary polynucleotide sequence) Is gradually increased until binding at a signal-to-noise ratio of at least 5x higher. In this case, the non-target nucleic acid is N. From an organism other than Gonoloaeae, and in some embodiments, C.I. It is from Trachomatis. Examples of such non-target nucleic acids are eg AE01469 (Brucella suis 1330, chromosome I, compartment 155) and AE002435 (Neisseria meningitidis), serogroup B, strain MC58, compartment 77) with a GenBank® accession number. Additional such sequences can be identified by those skilled in the art, for example in GenBank®.

  A test nucleic acid hybridizes to a probe as well as at least half as much as to a perfectly matched complementary target, i.e., a perfectly matched probe is a perfectly matched complementary Against the target, against the non-target nucleic acid AE01469 (Brucella suis 1330, chromosome I, compartment 155) or AE002435 (Neisseria meningitidis, serogroup B, strain MC58, compartment 77) When hybridized with a signal-to-noise ratio that is at least one half as high as the hybridization of the probe to the target under conditions that bind with a signal-to-noise ratio that is at least about 5x to 10x higher than that observed for hybridization Specifically hybridizes to nucleic acids Said to be soybeans.

  Ultra-high stringency hybridization and wash conditions result in a signal-to-noise ratio for probe binding to a perfectly matched complementary target nucleic acid, non-target nucleic acid AE01469 (Brucella suis 1330, chromosome I, compartment 155) or AE002435 (Neisseria meningitidis, serogroup B, strain MC58, compartment 77) of what is observed for hybridization and washing until a signal to noise ratio of at least 10 × higher. The stringency of conditions is increased. Under such conditions, a target nucleic acid that hybridizes to a probe with a signal-to-noise ratio of at least one-half that of a perfectly matched complementary target nucleic acid binds to the probe under ultra high stringency conditions. Said.

  Similarly, even higher levels of stringency can be determined by gradually increasing the stringency of the hybridization and / or wash conditions of the relevant hybridization assay. For example, the signal-to-noise ratio for binding of a probe to a perfectly matched complementary labeled nucleic acid is the non-target nucleic acid AE01469 (Brucella suis 1330, chromosome I, compartment 155) or AE002435 (Nyseria menis). A string of hybridization and wash conditions until at least 10x, 20x, 50x, 100x, or 500x or higher of what is observed for hybridization to Neisseria meningitidis, serogroup B, strain MC58, compartment 77) Those with increased genies can be identified. Under such conditions, a target nucleic acid that hybridizes to the probe with a signal-to-noise ratio of at least one-half that of a perfectly labeled complementary labeled nucleic acid binds to the probe under ultra high stringency conditions. Said.

  Detection of target nucleic acids that hybridize under high, ultra-high and ultra-high stringency conditions to the nucleic acids represented by SEQ ID NOs: 3-27 is a feature of the invention. Examples of such nucleic acids include those with one or several silent or conservative nucleic acid substitutions compared to a given nucleic acid sequence.

  The term “identity” or percent “identity” is measured in the context of two or more nucleic acid or polypeptide sequences, for example, using one of the sequence comparison algorithms available to those skilled in the art or by visual inspection. Two or more sequences or subsequences having the same or a certain percentage of the same amino acid residues or nucleotides when compared and aligned for maximum correspondence. A typical algorithm suitable for measuring percent sequence identity and sequence similarity is the BLAST program, for example, Altschul et al., “Basic local aligmnent search tool, each included as a reference. Local alignment research tool) ", J. Mol. Biol., 1990, 215, p. 403-410, Gish et al., “Identification of protein coding regions by database similarity search”, Nature Genet., 1993, Vol. 3, p. 266-272, Madden et al., “Applications of network BLAST server”, Meth. Enzymol., 1996, Vol. 266, p. 131-141, Altschul et al., “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res. 1997, Vol. 25, p. 3389-3402 and Zhang et al., “PowerBLAST: A new network BLAST application for interactive or automated sequence analysis and annotation”. Genome Res., 1997, Vol. 7, p. 649-656. Many other optimal alignment algorithms are also known in the art and are optionally used to measure percent sequence identity.

  The phrase “in solution” refers to assay or reaction conditions in which the components of the assay or reaction are not attached to the solid support and are present in the liquid medium. Typical liquid media include aqueous and organic solutions. For example, one assay of the present invention uses oligonucleotide probes as described in N.P. Gonorrhoeae nucleic acid and N.I. Incubating in solution with a gonoroae nucleic acid amplicon to allow hybridization to occur.

  The term “insert” in the context of a nucleic acid sequence refers to a change in which at least one nucleotide is added to a nucleic acid sequence, for example at the 5′-terminus, 3′-terminus, and / or an internal position of the nucleic acid sequence. Point to.

  “Label” refers to a component that can be attached (covalently or non-covalently) to or attached to a molecule, which can be a molecule or a labeled molecule with it. Information (eg, information about the molecule, description, identification, etc.) can be provided or provided for another molecule that interacts (eg, hybridizes, etc.). Representative labels include fluorescent labels (including quenchers or absorbers), weak fluorescent labels, non-fluorescent labels, colorimetric labels, chemiluminescent labels, bioluminescent labels, radioactive labels, mass modifying groups, antibodies, antigens , Biotin, haptens, enzymes (eg, peroxidase, phosphatase, etc.), and the like.

  A “linker” is a compound or substituent that is covalently or non-covalently attached to another component, eg, a nucleic acid, oligonucleotide probe, primer nucleic acid, amplicon, solid support, or the like. Refers to the chemical component to be attached. For example, a linker is optionally used to attach the oligonucleotide to a solid support (eg, a linear array or other logic probe array). Illustratively, the linker optionally attaches a label (eg, fluorescent dye, radioisotope, etc.) to the oligonucleotide probe, primer nucleic acid, or the like. Linkers are typically at least bifunctional chemical components, and in certain embodiments they comprise a detectable attachment that is cleaved by, for example, heat, enzymes, chemicals, electromagnetic radiation, and the like. It is possible to release the substance or the compound, for example from a solid support. Careful selection of the linker allows the cleavage to be performed under conditions suitable for the stability of the compound and the assay method. Usually, the linker does not have any specific biological activity other than, for example, by joining chemical species, or retaining some minimum distance or other spatial correlation between such species. However, the linker components may be selected to affect several properties of the linked species, such as the three-dimensional structure, net charge, hydrophobicity, etc. Exemplary linkers include, for example, oligopeptides, oligonucleotides, oligopolyamides, oligoethyleneglycerols, oligoacrylamides, alkyl chains, or the like. Additional descriptions of linker molecules can be found in, for example, Hermanson, “Bioconjugate Techniques”, Elsevier Science (1996), all contained in the references. Lytle et al., Nucleic Acids Res., 1996, 24, 14, p. 2793, Shchepino et al., Nucleosides, Nucleotides & Nucleic Acids, 2001, Volume 20, p. 369, Doronina et al., Nucleosides, Nucleotides & Nucleic Acids, 2001, Volume 20, p. 1007, Trawick et al., Bioconjugate Chem, 2001, Vol. 12, p. 900, Olejnik et al., Methods in Enzymology, 1998, Vol. 291, p. 135, and Pljevaljcic et al., J. Am. Chem. Soc., 2003, 125, 12, p. 3486.

  A “mass modifying” group modifies the mass of a molecule typically comprising the group, typically measured as a dalton in terms of molecular weight. For example, a mass modifying group that improves discrimination between at least two nucleic acids with a single base difference in size or sequence may be used to facilitate sequencing using, for example, molecular weight measurements. Is possible.

  “Mixture” refers to a combination of two or more different ingredients. “Reaction mixture” refers to a mixture comprising molecules that can participate in and / or promote a given reaction. “Amplification reaction mixture” refers to a solution containing the reagents necessary to perform an amplification reaction, typically containing a primer, a thermostable DNA polymerase, dNTPs, and a divalent metal cation in a suitable buffer. To do. A reaction mixture is called complete if it contains all the reagents necessary to carry out the reaction, and incomplete if it contains only a subset of the necessary reagents. For those skilled in the art, the reaction components are routinely separated, each containing a subset of the total components, for convenience, storage stability reasons, or to allow adjustment of component concentration depending on the application. It will be appreciated that it is stored as a solution and that the reaction components are combined prior to the reaction to create a complete reaction mixture. Furthermore, those skilled in the art will appreciate that the reaction mixture is packaged separately for commercialization, and that useful commercial kits may contain any subset of reaction components including the modified primers of the present invention. It will be understood.

  “Modified primer nucleic acid” refers to a primer nucleic acid comprising a component or nucleotide sequence that confers desired properties to the primer nucleic acid. In some embodiments, for example, a modified primer nucleic acid comprises a “nucleic acid amplification specificity-altering modification,” which, for example, reduces non-specific nucleic acid amplification (eg, minimizes primer dimer formation, or Otherwise), increasing the yield of the intended target amplicon and / or doing the same. Examples of nucleic acid amplification specificity-modifying modifications include, for example, “MODIFIED NUCLEIC ACID AMPLIFICATION PRIMERS” issued on December 14, 1999 to Will, which is included as a reference. In U.S. Pat. No. 6,001,611. Another exemplary primer nucleic acid modification is a “restriction site linker modification” in which a nucleotide sequence comprising a selected restriction site is attached to the 5′-end of the primer nucleic acid, for example. A restriction site linker is typically attached to the primer nucleic acid to facilitate subsequent amplicon cloning, or the like.

  “Component” or “group” refers to one of the moieties into which something, such as a molecule, is split (eg, a functional group, substituent, or the like). For example, the oligonucleotide probe optionally comprises a quencher component, a label component, or the like.

  The term “Neisseria gonorrhoeae”, “N. gonorrhoeae”, or “NG” refers to the bacterial species of the genus Neisseria, Gonorrhoeae. For example, Schoolnik (edition), “Pathogenic Neisseriae: Proceedings of the fourth International Symposium”, October 21-25, 1984, included as a reference. See Asilomar, California, American Microbiological Society, 1986. N. Gonoloae and C.I. A further general description of Trachomatis can be found in, for example, Stratus and Westran, “Clinical Bacteriology”, ASM Press & Manson Publishing, each included as a reference. ), 2003, Persing et al., “Molecular Microbiology: Diagnostic Principles and Practice”, ASM Press, 2003, by Murray, “Manual of Clinical Microbiology” (Clinical Microbiology Manual) ”, 8th edition, ASM Press, 2003. In addition, stdgen. lanl. See also the Neisseria Gonoeroe database on gov on the World Wide Web.

  The term “Neisseria gonorrhoeae nucleic acid” or “N. gonorrhoeae nucleic acid” refers to a nucleic acid (and / or its amplicon) derived from or isolated from Neisseria gonorrhoeae.

  The term “nucleic acid” refers to nucleotides (eg, ribonucleotides, deoxyribonucleotides, dideoxynucleotides, etc.) and polymers comprising such nucleotides covalently linked in a linear or branched fashion. Representative nucleic acids are deoxyribonucleic acid (DNA), ribonucleic acid (RNA), DNA-RNA hybrid, oligonucleotide, polynucleotide, gene, cDNA, aptamer, antisense nucleic acid, interfering RNA (RNAi), molecular beacon, nucleic acid probe , Peptide nucleic acids (PNA), locked nucleic acids (LNA®), PNA-RNA conjugates, LNA®-DNA conjugates, LNA®-RNA conjugates, and the like.

  Nucleic acids are typically single-stranded or double-stranded and usually contain phosphodiester linkages, but in some cases include nucleic acid analogs as outlined herein. It may have a modified skeleton, for example, without limitation, phosphoramides (each included by reference, Beaucage et al., Tetrahedron, 1993, 49, 10 No., p. 1925, and references therein; Letsinger, J. Org. Chem., 1970, 35, p. 3800; Sprinzl et al., Eur. J. Biochem., 1977 81, p. 579; Letsinger et al., Nucl. Acids Res., 1986, 14, p. 3487; Sawai et al., Chem. Lett., 1984, 805. ; J. Am. Che, by Letsinger et al. m. Soc., 1988, 110, p. 4470; and Pauwels et al., Chemica Scripta, 1986, 26, p. 1419), phosphorothioate (both included as references). Mag et al., Nucleic Acids Res., 1991, 19, p. 1437; and US Pat. No. 5,644,048), phosphorodithioate (included as a reference) Briu et al., J. Am. Chem. Soc., 1989, 111, p. 2321), O-methyl phosphoramidite linkage (included as a reference, by Eckstein). "Oligonucleotides and Analogues: A Practical Approach" (see Oxford University Press, 1992), and peptide nucleic acid backbone and Binding (Egholm, J. Am. Chem. Soc., 1992, 114, p. 1895, each included as a reference); Meier et al., Chem. Int. Ed. Engl., 1992, 31, p. 1008; Nielsen, Nature, 1993, 365, p. 566; and Carlsson et al., Nature, 1996, 380, p. 207). Other analog nucleic acids include positively charged scaffolds (Denpcy et al., Proc. Natl. Acad. Sci. USA, 1995, 92, p. 6097, included as a reference); Nonionic skeletons (US Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141, and 4,469, each included as a reference) 863; Angew, Chem. Intl. Ed. English, 1991, volume 30, p.423; Letsinger et al., J. Am. Chem. Soc., 1988, volume 110. 4470; Letsinger et al., Nucleoside & Nucleotide, 1994, 13, p. 1597; edited by YS Sanghvi & P. Dan Cook, “Carbohydrate Modifications in Antisense Research”. Sugar in antisense research ASC Symposium Series 580, Chapters 2 and 3; Mesmaeker et al., Bioorganic & Medicinal Chem. Lett., 1994, Vol. 4, p. 395; Jeffs et al. Biomolecular NMR, 1994, 34, p. 17; and Tetrahedron Lett., 1996, 37, p. 743) and US Pat. Nos. 5,235,033 and 5,034,506, And those described in YS Sanghvi and P. Dan Cook, “Carbohydrate Modifications in Antisense Research”, ASC Symposium Series 580, Chapters 6 and 7 Including a non-ribose skeleton. Nucleic acids containing one or more carbocyclic saccharides are also included within the definition of nucleic acids (included by reference, Jenkins et al., Chem. Soc. Rev., 1995, p. 169-176). Some nucleic acid analogs are also described, for example, by Rawls, C & E News, June 2, 1997, p. 35. Such modifications of the ribose-phosphate backbone may be performed to facilitate the addition of additional components such as labels or to alter the stability and half-life of such molecules in the physiological environment.

  In addition to these naturally occurring heterocycles typically found in nucleic acids (eg, adenine, guanine, thymine, cytosine, and uracil), nucleic acid analogs also contain non-naturally occurring heterocyclic or modified bases Many of which are described herein or otherwise referenced. In particular, many non-naturally occurring bases are further described in, for example, Seela et al., Helv. Chim. Acta, 1991, vol. 74, p. 1790, Grein et al., Bioorg. Med. Chem. Lett., 1994, Volume 4, p. 971-976, and Seela et al., Helv. Chim. Acta, 1999, vol. 82, p. 1640. To further illustrate, certain bases used in nucleotides that act as modifiers of melting point (Tm) are optionally included. For example, some of these include 7-deazapurine (eg, 7-deazaguanine, 7-deazaadenine, etc.), pyrazolo [3,4-d] pyrimidine, propynyl-dN (eg, propynyl-dU, propynyl-dC, etc.), and Other similar items are included. For example, “SYNTHESIS OF 7-DEAZA-2′-DEOXYGUANOSINE NUCLEOTIDES (7-deaza-2′-deoxyguanosine nucleotides) published on November 23, 1999 to Seela et al. U.S. Pat. No. 5,990,303. Other representative heterocyclic bases are, for example, hypoxanthine, inosine, xanthine; 8-aminopurine, 2,6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine, inosine, and xanthine. -Aza derivatives; 7-deaza-8-aza derivatives of adenine, guanine, 2-aminopurine, 2,6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine, inosine, and xanthine; 6-azacytosine; 5-fluorocytosine; 5-chlorocytosine; 5-iodocytosine; 5-bromocytosine; 5-methylcytosine; 5-propynylcytosine; 5-bromovinyluracil; 5-fluorouracil; 5-chlorouracil, 5-iodouracil; 5-bromouracil; 5-trifluoromethyluracil; 5-methoxy Methyl uracil; 5-ethynyl uracil; 5-propynyl uracil, and other like.

  Examples of modified bases and nucleotides are also US, entitled “OLIGONUCLEOTIDES CONTAINING 5-PROPYNYL PYRIMIDINES” published on 16 January 1996 to Froehler et al. Patent No. 5,484,908, “ENHANCED TRIPLE-HELIX AND DOUBLE-HELIX FORMATION WITH OLIGOMERS CONTAINING MODIFIED PYRIMIDINES” issued on July 8, 1997 to Froehler et al. “METHODS OF USING OLIGOMERS CONTAINING MODIFIED PYRIMIDINES” issued on November 3, 1998 to Froehler et al., US Pat. No. 5,645,985 entitled US Pat. No. 5, entitled “Use of Oligomers Containing Modified Pyrimidines” No. 830,653, US patent number “SYNTHESIS OF [2.2.1] BICYCLO NUCLEOSIDES” (synthesized [2.2.1] bicyclonucleoside) issued to Kochkine et al. On October 28, 2003. No. 6,639,059, “ONE STEP SAMPLE PREPARATION AND DETECTION OF NUCLEIC ACIDS IN COMPLEX BIOLOGICAL SAMPLES” issued on October 16, 2001 to Skouv et al. US Pat. No. 6,303,315 entitled “Detection of Nucleic Acids” and “SYNTHESIS OF [2.2.1] BICYCLO NUCLEOSIDES ([2.2”) published on May 15, 2003 to Kochkine et al. .1] Synthesis of Bicyclonucleosides) is also described in US 2003/0092905, each of which is incorporated by reference. ing.

  The term “nucleic acid detection reagent” is SEQ ID NO: 1, SEQ ID NO: 2, a variant thereof substantially identical and having at least 90% sequence identity with one of SEQ ID NO: 1 or 2. Or a nucleic acid comprising SEQ ID NO: 1, SEQ ID NO: 2, or the complement of said variant, detectably bind (eg, hydrogen bonding in antibody-antigen recognition in nucleic acid hybridization, and the like) Or other types of binding interactions). For example, nucleic acids (for example, probe nucleic acids, primer nucleic acids, etc.) comprising sequences selected from SEQ ID NOs: 3-27 or their complements specifically bind to nucleic acids having these sequences. Another exemplary nucleic acid detection reagent is SEQ ID NO: 1, SEQ ID NO: 2, a variant thereof substantially identical and having at least 90% sequence identity with one of SEQ ID NO: 1 or 2. Or a sequence-specific antibody that specifically binds to a nucleic acid comprising SEQ ID NO: 1, SEQ ID NO: 2, or a complement of said variant.

  “Nucleotide” refers to an ester of a nucleoside, eg, a phosphate ester of a nucleoside. For example, the nucleotide can include 1, 2, 3, or more phosphate groups covalently linked to the 5 'position of the sugar component of the nucleoside.

  “Nucleotide uptake biocatalyst” refers to a catalyst that catalyzes the incorporation of nucleotides into a nucleic acid. The nucleotide uptake biocatalyst is typically an enzyme. An “enzyme” is a catalyst based on proteins and / or nucleic acids, and acts to reduce the activation energy of chemical reactions involving other compounds or “substrates”. “Nucleotide uptake enzyme” refers to an enzyme that catalyzes the incorporation of nucleotides into a nucleic acid, for example during nucleic acid amplification or the like. Exemplary nucleotide uptake enzymes include, for example, polymerase, terminal translase, reverse transcriptase, telomerase, polynucleotide phosphorylase.

  “Oligonucleotide” refers to a nucleic acid comprising at least two nucleic acid monomer components (eg, nucleotides), typically more than three monomer units, and more typically more than ten monomer units. The exact size of the oligonucleotide usually depends on a variety of factors, including the ultimate function or use of the nucleotide. Oligonucleotides include, but are not limited to, isolation of existing or native sequences, DNA replication or amplification, reverse transcription, cloning of appropriate sequences and restriction enzyme digestion, or Narang et al., Meth. Enzymol., 1979. Year 68, p. 90-99 phosphate triester method; Brown et al., Meth. Enzymol., 1979, Vol. 68, p. 109-151 phosphodiester method; Beaucage et al., Tetrahedron Lett., 1981, Vol. 22, p. 1859-1862, diethylformolamidite method; Matteucci et al., J. Am. Chem. Soc., 1981, vol. 103, p. 3185-3191 triester method; automated synthesis method; or direct chemical synthesis by methods such as solid support method of US Pat. No. 4,458,066, or methods known in the art. It is optionally prepared by any suitable method, including. All these references are included for reference.

  The term “oligonucleotide probe”, “probe nucleic acid”, or “probe” is a labeled or unlabeled oligonucleotide capable of selectively hybridizing to a target nucleic acid under appropriate conditions. Point to. Typically, a probe is a specific target sequence contained in a nucleic acid sample (eg, SEQ ID NO: 1, SEQ ID NO: 2, a substantially identical variant thereof and one of SEQ ID NO: 1 or 2). A variant having at least 90% sequence identity, or N. gonorrhoeae nucleic acid comprising SEQ ID NO: 1, SEQ ID NO: 2 or the complement of said variant), C.I. Is sufficiently complementary to, eg, a Trachomatis nucleic acid sequence) to form a stable hybridization duplex with the target sequence under selected hybridization conditions such as, but not limited to, stringent hybridization conditions. Hybridization assays performed under sufficiently stringent hybridization conditions using probes allow selective detection of specific target sequences. The term “hybridizing region” refers to a region of a nucleic acid that is exactly or substantially complementary to a target nucleic acid and thus hybridizes to the target. For use in hybridization assays for the identification of single nucleotides in a sequence, the hybridizing region is typically about 8 to about 100 nucleotides in length. Although the hybridizing region usually refers to the entire oligonucleotide, the probe may include additional nucleotide sequences, for example to provide a site for attaching the probe sequence to a solid support or the like, Functions as a linker binding site. In some embodiments, the oligonucleotide probes of the invention comprise one or more labels (eg, reporter dyes, quencher components, etc.), such as FRET probes, molecular beacons, or the like, which also And can be used to detect hybridization between the target nucleic acid and the target nucleic acid in the sample. In some embodiments, the hybridizing region of the oligonucleotide probe is completely complementary to the target sequence. Usually, however, perfect complementarity is not required and stable duplexes may contain mismatched or unpaired bases. Changes in stringent conditions may be necessary to allow stable hybridization duplexes with one or more base pair mismatches or unpaired bases. Included as a reference, Sambrook et al., “Molecular Cloning: A Laboratory Manual” 3rd edition, Cold Spring Harbor Laboratory Press, New York The State, Cold Spring Harbor, 2001, provides guidelines on appropriate changes. The stability of the target / probe duplex depends on many variables including the length of the oligonucleotide, the base composition and sequence of the oligonucleotide, temperature, and ionic strength. One skilled in the art will recognize that the exact complement of a given probe is usually useful as a probe as well. SEQ ID NO: 1, SEQ ID NO: 2, a variant thereof substantially the same and having at least 90% sequence identity with one of SEQ ID NO: 1 or 2, or SEQ ID NO: 1, sequence No. 2, or N. having a sequence consisting of the complement of the mutant. An exemplary probe of the present invention that binds to a Gonorrhoeae nucleic acid comprises a sequence selected from SEQ ID NOs: 3-27 and its complement. One skilled in the art will also recognize that in certain embodiments, probe nucleic acids can also be used as primer nucleic acids.

  A “primer nucleic acid” or “primer” is a template nucleic acid (eg, N. gonorrhoeae nucleic acid, SEQ ID NO: 1, SEQ ID NO: 2, a substantially identical variant thereof, of SEQ ID NO: 1 or 2 Or a variant having at least 90% sequence identity with one, or a nucleic acid comprising SEQ ID NO: 1, SEQ ID NO: 2, or the complement of said variant, C. trachomatis nucleic acid, etc.) A nucleic acid that is capable of extending or extending a strand under suitable reaction conditions, eg, using a nucleotide-incorporating biocatalyst such as a polymerase. Primer nucleic acids are typically natural or synthetic oligonucleotides (eg, single-stranded oligodeoxynucleotides, etc.). Other primer nucleic acid lengths are optionally utilized, but they typically comprise a hybridizing region that ranges from about 8 to about 100 nucleotides in length. Short primer nucleic acids typically utilize the colder temperature to make template N.I. Gonoloae or C.I. It forms a sufficiently stable hybrid complex with the Trachomatis nucleic acid. Template N. Gonoloae or C.I. A primer nucleic acid that is at least partially complementary to a partial sequence of a Trachomatis nucleic acid is typically sufficient to hybridize with a template due to extension. A primer nucleic acid can be labeled if desired, for example, by incorporating a label detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other techniques. is there. Illustratively, useful labels include radioisotopes, fluorescent dyes, high electron density reagents, enzymes (usually used in ELISA), biotin, or haptens and antisera or monoclonal antibodies against them. Contains a protein. Many of these and other labels are further described herein and / or are known in the art. SEQ ID NO: 1, SEQ ID NO: 2, a variant thereof that is substantially identical and having at least 90% sequence identity with one of SEQ ID NO: 1 or 2, or SEQ ID NO: 1, sequence No. 2 or N. having a sequence consisting of the complement of the mutant. An exemplary primer of the present invention that binds to a Gonorrhoeae nucleic acid comprises a sequence selected from SEQ ID NOs: 3-27 and its complement. One skilled in the art will recognize that in certain embodiments, primer nucleic acids can also be used as probe nucleic acids.

  A “quencher component” or “quencher” reduces the emission of detectable luminescence emitted by the source that otherwise emitted this luminescence, eg, fluorescence or luminescent radiation, and / or It refers to a component that can be reduced. The quencher typically reduces the detectable emission emitted by the source by at least 50%, typically at least 80%, more typically at least 90%. A typical quencher is, for example, “OLIGONUCLEOTIDE PROBES BEARING QUENCHABLE FLUORESCENT LABELS, AND METHODS OF USE THEREOF” published on October 15, 2002 to Horn et al. And its use) is provided in US Pat. No. 6,465,175, which is incorporated by reference.

  The term “sample” includes, but is not limited to, tissue or fluid isolated from one or more subjects or individuals, in vitro cell culture components, and clinical samples. Gonoloae and / or C.I. Refers to any substance that contains or is thought to contain a Trachomatis nucleic acid. Typical samples are blood, plasma, serum, urine, joint fluid, semen, seminal plasma, prostate fluid, vaginal fluid, cervical fluid, uterine fluid, cervical abrasion, amniotic fluid, anal scrape, mucus, sputum, Includes organizations, and the like.

  The term “sample from a subject” is obtained from a subject regardless of whether the sample undergoes one or more processing steps (eg, cell lysis, debris removal, stabilization, etc.) prior to analysis. Sample. Illustratively, a sample can be taken from a subject by abrasion, venipuncture, swabbing, biopsy, or techniques known in the art.

  The term “selectively binds” or “selective binding” in the context of a nucleic acid detection reagent is SEQ ID NO: 1, SEQ ID NO: 2, a substantially identical variant thereof, SEQ ID NO: 1 or 2 A variant having at least 90% sequence identity with one of the above, or a sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, or a complement of said variant. For a Gonorrhoeae nucleic acid, it refers to a nucleic acid detection reagent that binds more than the nucleic acid detection reagent binds to nucleic acids from at least three organisms each selected from Tables X and XI under the same hybridization conditions.

  The term “selectively detect” is SEQ ID NO: 1, SEQ ID NO: 2, a variant thereof substantially identical and having at least 90% sequence identity with one of SEQ ID NO: 1 or 2. Or a mutant having a sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, or the complement of the mutant. It refers to the ability to detect Gonorrhoeae nucleic acids more than nucleic acids from other organisms.

  “Selectively hybridize” or “selective hybridization” means that a nucleic acid hybridizes to a specific nucleic acid target sequence to a detectably higher degree than its hybridization to a non-target nucleic acid sequence. It happens when you do. Sequences that selectively hybridize to each other at least 50%, or 60%, or 70%, or 80%, or 90% sequence identity or higher, eg, typically 95-100 % Sequence identity (ie complementarity).

  The “sequence” of a nucleic acid refers to the order and identity of the nucleotides within the nucleic acid. The sequence is typically read in the 5 'to 3' direction.

  “Sequence-specific antibody” is a variant that is substantially the same as SEQ ID NO: 1, SEQ ID NO: 2, and has at least 90% sequence identity with one of SEQ ID NO: 1 or 2 Or an antibody that detectably binds to a nucleic acid having a sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, or the complement of the mutant.

  A “sequencing reaction” refers to a reaction that includes, for example, the use of terminator nucleotides that are designed to elucidate the nucleotide sequence in a given nucleic acid.

  A “set” refers to a population of at least two things. For example, the set may include 2, 3, 4, 5, 10, 20, 50, 100, 1000, or other numbers of molecules or sequence types. For example, one aspect of the present invention includes a reaction mixture having a set of amplicons. “Subset” refers to any part of a set.

  "Solid support" refers to a solid material that can be coated with or otherwise attached to a chemical moiety such as an oligonucleotide probe or the like. Exemplary solid supports include plates, beads, microbeads, tubes, fibers, whiskers, combs, hybridization chips (GeneChip® probe arrays (Affymetrix, Inc., California, USA, Including microarray substrates, such as those used in Santa Clara), membranes, single crystals, ceramic layers, self-assembled monolayers, and the like.

  An oligonucleotide probe is targeted if the number of mismatches that exist between the oligonucleotide and the target nucleic acid is less than the number of mismatches that exist between the oligonucleotide and the non-target nucleic acid that may be present in the sample. “Specific” to the nucleic acid. Hybridization conditions can be selected under which stable duplexes are only present if the number of mismatches present does not exceed the number of mismatches present between the oligonucleotide and the target sequence. Is formed. Under such conditions, the target-specific oligonucleotide is capable of forming a stable duplex only with the target sequence. Thus, the use of target-specific primers under appropriately stringent amplification conditions allows for specific amplification of those sequences containing the target primer binding site. Similarly, the use of target specific probes under appropriately stringent hybridization conditions allows for the detection of specific target sequences.

  “Subject” refers to an organism. Typically, the organism is a mammalian organism, particularly a human organism. In some embodiments, for example, the subject is a patient suspected of having NG and / or CT infection.

  “Partial sequence” or “segment” refers to any portion of an entire nucleic acid sequence.

  A “substantially identical variant” in the context of a nucleic acid or polypeptide is when compared and aligned for maximum correspondence, as measured, for example, using a sequence comparison algorithm or by visual inspection. , Refers to two or more sequences having at least 85%, typically at least 90%, more typically at least 95% nucleotide or sequence identity to each other. Substantial identity usually spans a sequence region that is at least about 15 nucleotides or amino acids in length, more typically over a sequence region that is at least about 20 nucleotides or amino acids in length, and even more typically at least about 25 nucleotides. Or it exists over a sequence region that is amino acid long. In some embodiments, for example, the sequences are substantially identical over the entire length of the compared nucleic acid or polypeptide.

  The term “substitution” in the context of a nucleic acid sequence refers to a change in which at least one of the nucleic acid sequences is replaced by another nucleotide.

  The terms “target sequence”, “target region”, and “target nucleic acid” refer to a region of a nucleic acid that is to be amplified, detected, or otherwise analyzed.

  A “terminator nucleotide” refers to a nucleotide that prevents further extension of a nucleic acid upon incorporation into a nucleic acid, eg, by at least one nucleotide incorporation biocatalyst.

  A “thermostable enzyme” refers to an enzyme that is stable to heat, heat resistant, and retains sufficient catalytic activity when exposed to elevated temperatures for a selected period of time. For example, a thermostable polymerase retains sufficient activity to effect subsequent primer extension when exposed to high temperatures for the period necessary to cause denaturation of double-stranded nucleic acids. The heating conditions necessary for nucleic acid denaturation are known in the art and are typified by US Pat. Nos. 4,683,202 and 4,683,195, both of which are incorporated by reference. As used herein, thermostable polymerases are typically suitable for use in temperature cycling reactions such as the polymerase chain reaction (“PCR”). For thermostable polymerases, the enzyme activity is in an appropriate manner to form a primer extension product that is complementary to a template nucleic acid (eg, a selected subsequence of N. gonorrhoeae or C. trachomatis genome). Refers to catalysis for a combination of nucleotides.

II. Overview The present invention relates to the selective detection of Neisseria gonorrhoeae. In particular, N.I. Based on a novel detection strategy utilizing at least two target regions of the Gonorrhoeae genome, using the methods and reagents described in this invention, N. A Gonorrhoeae infection can be diagnosed. Each of these target regions is Has multiple copies in the Gonorrhoeae genome. Therefore, this is typically compared to techniques that target a single copy region in the genome as compared to N. Facilitates detection of Gonoloaeae. In addition, the nucleic acid detection reagents described herein are typically N. cerevisiae under selected assay conditions. Detectably binds to nucleotide sequences that are present in Gonorrhoeae but not in other species, thereby, for example, minimizing the occurrence of false positives. This specificity is illustrated by way of example in, for example, FIGS. 5-7, 9, and 10 and the related description in the examples provided below.

  Illustratively, certain methods of the invention contact a nucleic acid detection reagent with nucleic acid in or from a sample derived from a subject (eg, a human patient suspected of having N. gonorrhoeae infection). Including. In some embodiments, the target region of the nucleic acid in the sample is amplified before or simultaneously with the nucleic acid detection reagent. The nucleic acid detection reagent is SEQ ID NO: 1, SEQ ID NO: 2, a variant thereof that is substantially identical and having at least 90% sequence identity with one of SEQ ID NO: 1 or 2, or It binds detectably to a nucleic acid having a sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, or the complement of the mutant. As further described below, SEQ ID NO: 1 and SEQ ID NO: 2 are targeted to N. It is a consensus sequence corresponding to two regions of Gonoloae. These methods also monitor binding between the nucleic acid and / or amplicon and the nucleic acid detection reagent (eg, at one time point, at a number of separate time points, continuously over a selected time, etc.). For example, to diagnose the patient from whom the sample was derived, to monitor the course of treatment for a patient diagnosed with Neisseria gonorrhoeae infection, and / or for the like, Neisseria gonorrhoeae is Determine if it exists.

  In some embodiments, these methods further comprise contacting the target region nucleic acid and / or amplicon with an additional nucleic acid detection reagent that detectably binds to the Chlamydia trachomatis nucleic acid. In these embodiments, the method also includes monitoring binding between the nucleic acid and / or amplicon and the additional nucleic acid detection reagent to determine whether Chlamydia trachomatis is also present in the sample. Optionally, these methods also use an additional sample (eg, from the same subject) to treat the course of a subject diagnosed with, for example, Neisseria gonorrhoeae and / or Chlamydia trachomatis infection, Repeated one or more times to monitor the recurrence of infection and / or the like.

  In another method of the present invention, a nucleic acid derived from a sample is contacted with at least one first pair of primer nucleic acids comprising at least one nucleic acid selected from the group consisting of SEQ ID NOs: 3 to 27 or a complement thereof. Or incubating. As further described below, SEQ ID NOs: 3-27 are oligonucleotides comprising a partial sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In addition, these methods also include detecting amplicons during or after the amplification reaction to determine whether Neisseria gonorrhoeae is present in the sample. Optionally, these methods further comprise contacting the nucleic acid from the sample with at least one second pair of primer nucleic acids that are at least partially complementary to the Chlamydia trachomatis nucleic acid, and an amplification reaction is performed. During or after, detecting additional amplicons to determine if Chlamydia trachomatis is present in the sample. These methods are also optionally repeated at the selected time.

  In addition to compositions and reaction mixtures, the present invention also relates to kits and systems for detecting these pathogenic agents, and related computers and computer-readable media.

III. Nucleic Acid Detection Reagent The nucleic acid detection reagent of the present invention includes various embodiments such as a probe nucleic acid, a primer nucleic acid, and a sequence-specific antibody. These nucleic acid detection reagent target repeats 130 (also referred to herein as “NGDR9”) A direct repeat of 806 base pairs in the Gonorrhoeae genome (repetitive in the same direction) and is thought to encode a protein. N. The Gonorrhoeae genome contains two copies of NGDR9, one localized at nucleotide positions 458182 to 458988 and the other localized at nucleotide positions 1586504 to 1587310. The consensus sequence for NGDR9 corresponds to SEQ ID NO: 1, which is shown in Table 1. Although only one strand of the NGDR9 locus is shown in Table 1, one of ordinary skill in the art will recognize that SEQ ID NO: 1 identifies a region of a double-stranded genomic nucleic acid and that the sequence information provided provides both strands. It will be appreciated that the array is fully specified.

  Illustrative examples are nucleic acid detection reagents comprising SEQ ID NOs: 3-12, 17-20, 24-26, or complements thereof, target NGDR9 or complements thereof. SEQ ID NOs: 3-12, 17-20, and 24-26 are shown in Table 2.

  Another nucleic acid detection reagent of the present invention, target repeat 116 (also referred to as “NGDR33”) A direct repeat of 1142 base pairs in the Gonorrhoeae genome that encodes a polypeptide. N. The Gonorrhoeae genome contains two copies of NGDR33, one localized at nucleotide positions 491768-492910 and the other localized at nucleotide positions 16066987-160129. The consensus sequence for NGDR33 corresponds to SEQ ID NO: 2, which is shown in Table 3. Although only one strand of the NGDR33 locus is shown in Table 3, one of ordinary skill in the art knows that SEQ ID NO: 2 identifies a region of a double-stranded genomic nucleic acid and that the sequence information specified indicates both strands. It will be appreciated that the array is fully specified.

  For example, a nucleic acid detection reagent containing SEQ ID NOs: 13-16, 21-23, 27, or a complement thereof, target NGDR33 or a complement thereof. SEQ ID NOs: 13-16, 21-23, and 27 are shown in Table 4.

  In certain embodiments where NGDR9 is the target, the probes and / or primers are optionally of SEQ ID NO: 1: 259, 260, 262, 264, 265, 266, 268, 269, 273, 275, 276, 277, 279 , 297, 298, 300, 301, 302, 303, 304, 305, 306, 308, 313, 314, 315, 316, 317, 318, 320, 321, 325, 326, 428, 429, 431, 432, 433 Detectably binds to a nucleic acid segment comprising one or more nucleotide positions selected from the group consisting of 434, 435, 440, 441, and 447. These nucleotide positions are highlighted and underlined in Table 5, with the sequence of Brucella suis 1330, chromosome I, compartment 155 (GenBank® accession number AE014469) and Several representative mismatches, indicating that the sequence of NGDR9 and B. Identified in alignment with Swiss 1330 chromosome I, compartment 155. Another mismatch with this sequence from the Brucella Swiss genome is illustrated in FIG. B. This sequence of the Swiss genome has a higher level of identity with NGDR9 than sequences from other bacterial species. B. of the sequence of NGDR9. The alignment with the Swiss sequence is further described in the examples provided below.

  In some embodiments, NGDR33 is targeted and the probe and / or primer is optionally selected from SEQ ID NO: 2: 89, 90, 91, 92, 95, 98, 101, 105, 106, 107, 216, 217, 220, 222 223, 225, 233, 235, 236, 238, 335, 336, 337, 338, 339, 342, 345, 346 and a nucleic acid segment comprising one or more nucleotide positions selected from the group consisting of 351 On the other hand, it binds detectably. These nucleotide positions are highlighted and underlined in Table 6, Neisseria meningitidis serogroup B, strain MC58, compartment 77 (GenBank® accession number AE002435). ) Shows some representative mismatches with the sequence of NGDR33 and N. Identified in alignment with Meningitidis serogroup B, strain MC58, compartment 77. N. Another mismatch with this sequence from the Meningitidis genome is illustrated in FIG. N. This sequence of the Meningitidis genome has a higher level of identity with NGDR33 than sequences from other bacterial species. Of the sequence of NGDR33. Alignment with the Meningitidis sequence is further described in the examples provided below.

  As described above, in some embodiments of the present invention, the nucleic acid detection reagent comprises an oligonucleotide (eg, probe nucleic acid, primer nucleic acid, etc.). Although other lengths are optionally utilized, oligonucleotides typically are typically between about 8 and about 100 nucleotides in length, more typically between about 10 and about 75 nucleotides in length, and even more A sequence that is typically between about 12 and about 50 nucleotides in length, more typically between about 15 and about 35 nucleotides in length (eg, about 20, about 25, or about 30 nucleotides in length). Comprising. Methods for preparing oligonucleotides, such as nucleic acid synthesis, are further described below.

  To design an oligonucleotide (eg, a substantially identical variant of a nucleic acid having a sequence selected from SEQ ID NOs: 3-27 or its complement) that selectively binds to NGDR9 or NGDR33 by one skilled in the art. Various approaches are available and this oligonucleotide is It can be used to detect Gonorrhoeae. Illustratively, the DNAstar software package available from DNASTAR (Madison, Wis.) Can be used for sequence alignment. For example, NGDR9 and B.I. Switzerland, or NGDR33 and N.I. Nucleic acid sequences for Meningitidis can be uploaded to the DNAstar EditSeq program as individual files. Sequence file pairs (eg, NGDR9 and B. Switzerland) are opened in the DNAstar MegAlign alignment program and the ClustalW alignment method can be applied. The parameters used for ClustalW alignment are optionally software default settings. MegAlign typically does not provide an overview of percent identity between two sequences. This is usually calculated manually. From the alignment, for example, regions with less than 85% identity to each other are typically identified and oligonucleotide sequences within these regions can be selected. Many other sequence alignment algorithms and software packages are also optionally utilized. Sequence alignment algorithms are also described, for example, by Mount, “Bioinformatics: Sequence and Genome Analysis”, Cold Spring Harbor Laboratory Press, 2001, both of which are included as references. Also described in Durbin et al., “Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids”, Cambridge University Press, 1998. Has been.

  By way of further illustration, the optimal alignment of sequences for comparison is, for example, by Smith & Waterman, Adv. Appl. Math., 1981, Volume 2, each included as a reference. , P. According to the 482 local homology algorithm, Needleman & Wunsch, J. Mol. Biol., 1970, Vol. 48, p. 443 homology alignment algorithm, Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA, 1988, vol. 85, p. The search for 2444 similarity methods and the GAP of these algorithms (Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wis.) , BESTHIT, FASTA, TFASTA)) or by visual inspection.

  Another exemplary algorithm suitable for determining percent sequence identity is described, for example, by Altschul et al., J. Mol. Biol., 1990, 215, p. BLAST algorithm described in 403-410, which is included as a reference. Software for performing a version of BLAST analysis is available from ncbi. nlm. nih. Publicly available through the National Center for Biotechnology Information on the World Wide Web at gov /. The algorithm identifies short words of length W in the search sequence that, when aligned with words of the same length in the database sequence, match or satisfy a certain positive value threshold score T, It involves first identifying a high scoring sequence pair (HSP). T is referred to as the neighborhood word score threshold (Altschul et al., Supra). These first neighborhood food word hits act as seeds for initiating searches to find longer HSPs containing them. The word hit is then extended in both directions along each sequence as long as the cumulative alignment score can be increased. The cumulative score is calculated for the nucleotide sequence using the parameters M (reward for matched residue pair; usually> 0) and N (penalty score for mismatched residue; usually <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The extension of the word hits in each direction is: when the cumulative alignment score drops from its maximum achieved value to the quantity X, when the cumulative score falls below zero due to the alignment of one or more negative score residues, Or it is stopped when either sequence reaches the end. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses word length (W) 11, expectation (E) 10, reference value (cutoff) 100, M = 5, N = 4, and comparison of both strands by default. For amino acid sequences, the BLASTP program defaults to a word length (W) of 3, an expected value (E) of 10, and a BLOSUM62 scoring matrix (eg, included by reference, by Henikoff and Henikoff) Proc. Natl. Acad. Sci. USA, 1989, 89, p. 10915).

  In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (eg, Karlin & Artur (Karlin & Arthur, included as a reference)). Altschul), Proc. Nat'l. Acad. Sci. USA, 1993, 90, p. 5873-5787). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P (N)), which provides an indication of the probability that a match between two nucleotide sequences will occur by chance. For example, in a comparison of a test nucleic acid to a reference nucleic acid, if the minimum sum probability is less than about 0.1, or less than about 0.01, and / or less than about 0.001, the nucleic acid is similar to the reference sequence (and Therefore, it is considered to be homologous.

  An additional example of a useful sequence alignment algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive pairwise alignments. It can also plot a tree showing the clustering relationships that were used to create the alignment. PILEUP is described in Feng & Doolittle, J. Mol. Evol., 1987, vol. 35, p. Use the progressive alignment method simplification of 351-360. The method used is described by Higgins & Sharp, CABIOS, 1989, Vol. 5, p. Similar to the method described by 151-153. For example, the program can align up to 300 sequences with a maximum length of 5,000 characters. The multiple alignment method starts with a pair-wise alignment of the two most similar sequences and produces a cluster of the two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. A cluster of two sequences can be aligned by a simple extension of the pairwise alignment of two individual sequences. Final alignment is achieved by a series of progressive, pair-wise alignments. The program can also be used to plot a clustered relationship dendogram or tree display. The program is run by specifying a particular sequence and its amino acid or nucleotide coordinates for the region of sequence comparison.

  The probes and primers of the present invention optionally include one or more labels, which are further described below. In addition, the probes and primers are optionally modified nucleotides that change the melting temperature of the hybridization, restriction site linkers to facilitate amplicon cloning, modifying groups that increase the specificity of the nucleic acid amplification reaction, and / or the like Including various other modifications. For example, certain modified nucleotides that increase the melting point of nucleic acid hybridization are optionally included to allow the use of smaller probes, such as those involving between about 8 and about 14 nucleotides. Examples of these modified oligonucleotides include those having one or more LNA® monomers. Such nucleotide analogues are described, for example, in “SYNTHESIS OF [2.2.1] BICYCLO NUCLEOSIDES (synthesis of [2.2.1] bicyclonucleosides) published on October 28, 2003 to Kochkine et al. US Patent No. 6,639,059 entitled “ONE STEP SAMPLE PREPARATION AND DETECTION OF NUCLEIC ACIDS IN COMPLEX BIOLOGICAL SAMPLES” issued on October 16, 2001 to Skouv et al. US Pat. No. 6,303,315 entitled “Step Sample Preparation and Nucleic Acid Detection” and “SYNTHESIS OF [2.2.1] BICYCLO NUCLEOSIDES” published May 15, 2003 to Kochkine et al. Further described in US Patent Application Publication No. 2003/0092905 entitled [Synthesis of [2.2.1] bicyclonucleosides]. These are each included as references: Oligonucleotides comprising LNA® monomers are commercially available, for example, from Exiqon A / S (Vedbaek, Denmark). Probe and primer modifications are referred to herein, including those in the definitions set forth in the preceding sentence.

In certain embodiments, a nucleic acid detection reagent utilized as described herein is SEQ ID NO: 1, SEQ ID NO: 2, a substantially identical variant thereof, and one of SEQ ID NO: 1 or 2. A sequence-specific antibody using as a label a mutant having at least 90% sequence identity, or SEQ ID NO: 1, SEQ ID NO: 2, or the complement of the mutant. SEQ ID NO: 1 and SEQ ID NO: 2 are further described in the preamble and are provided in Tables 1 and 2, respectively. Antibodies suitable for use in these aspects of the invention may be prepared by routine methodology and / or by genetic engineering. Antibody fragments may also be genetically engineered, eg, from light and / or heavy chain variable regions, including hypervariable regions (V _H and V _L ), or from both V _H and V _L regions. Good. For example, the term “antibody” as used herein includes polyclonal and monoclonal antibodies, as well as biologically active fragments thereof, among other possibilities being “monovalent” antibodies (Grenier ( Glennie et al., Nature, 1982, 295, p. 712); included Fab ′ and F (ab ′) ₂ fragments, whether covalently or non-covalently aggregated. Fab protein; light or heavy chain only, typically variable heavy and light chain regions (V _H and V _L regions) to, more typically complementarity determining regions of the V _H and V _L region hypervariable regions (other (Known as CDRs); Fc proteins; “hybrid” antibodies capable of binding one or more antigens; constant-variable region chimeras; “complex” immunoglobulins with heavy and light chains of different origins; "Modified" with improved specificity and other characteristics, as prepared by quasi-recombinant techniques, by mutation techniques, or by other directed evolution techniques known in the art Contains antibodies.

  The utilized sequence specific antibodies described herein may be labeled or unlabeled. Suitable labels include, for example, radionuclides, enzymes, coenzymes, fluorescent dyes, chemiluminescent dyes, chromogens, enzyme substrates or cofactors, enzyme inhibitors, free radicals, and the like. Such labeled reagents may be used in a variety of well-known assays such as radioimmunoassays, enzyme immunoassays such as ELISA, fluorescent immunoassays, and the like. See, for example, US Pat. Nos. 3,766,162, 3,791,932; 3,817,837; and 4,233,402, each of which is incorporated by reference. Additional labels are further described herein.

  In some embodiments, transcribed RNA and / or translated proteins encoded by NGDR9 and NGDR33 are targeted for detection. Many techniques for detecting RNA and / or proteins are known in the art. For example, the probe and primer nucleic acids of the invention can be adapted for use in reverse transcription polymerase chain reaction (RT-PCR) for the detection of NGDR9 or NGDR33 transcripts. In addition, various electrophoretic assays (eg, SDS-PAGE or the like), immunoassays, mass spectrometry assays (eg, matrix-assisted laser desorption / ionization (MALDI) based analysis, surface enhanced laser desorption / ionization ( SELDI) based analysis, etc.), and / or other approaches can be used to detect proteins encoded by NGDR9 or NGDR33. Many of these and other suitable RNA and protein detection methods are described in the references cited herein.

  In practicing the present invention, many conventional techniques in molecular biology and recombinant DNA are optionally used. These techniques are well known, for example, FM Ausubel (eds.), “Current Protocols in Molecular Biology,” Volumes I, II, and III, 1997; Sunbrook ( Sambrook et al., “Molecular Cloning: A Laboratory Manual”, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001; Berger & Kimmel, “Guide to Molecular Cloning Techniques, Methods in Enzymology”, vol. 152, Academic Press, San Diego, Calif. ; Glover (DN Glover), "DNA Cloning: A Practical Approach (DNA cloning: practical approaches), Volumes I and II, 1985; edited by ML Gait, “Oligonucleotide Synthesis”, 1984; by Hames and Higgins, “ Nucleic Acid Hybridization, 1985; Hams & Higgins, Transcription and Translation, 1984; Freshney, Animal Cell Culture. Culture "), 1986;" Immobilized Cells and Enzymes ", IRL Publishing, 1986; by Perbal," A Practical Guide to Molecular Cloning ", 1984; “the series, Methods in Enzymology”, Academi CUPRES; edited by JH Miller & MP Calos, “Gene Transfer Vectors for Mammalian Cells”, Cold Spring Harbor Laboratory, 1987; edited by Wu & Grossman And Wu, Methods in Enzymology, Volumes 154 and 155, respectively, all of which are included as references.

IV. Sequence Variations Many nucleic acid and polypeptide sequences are within the scope of the present invention. Illustratively, FIG. 1 shows various N. cerevisiae sequences of the Neisseria gonorrhoeae direct repeat 9 (NGDR9) sequence. The sequence alignment is shown with the sequence of at least the amplicon part of the genomic DNA from the Gonorrhoeae strain. More specifically, five N.I. Genomic DNA from Gonorrhoeae strains (ie, NG strains 1117, 1120, 6346, 6359, and 6364) are amplified and sequenced using primer nucleic acids corresponding to DK101 (SEQ ID NO: 7) and DK102R (SEQ ID NO: 25). . The positions of the complements to oligonucleotides DK101 and DK102R (ie, DK102 (SEQ ID NO: 8)) and oligonucleotides NG519 (SEQ ID NO: 5) and NG514 (SEQ ID NO: 6) are in the sequence of NGDR9 shown in FIG. Underlined. In addition, the DR9 sequence and five N.P. Most or consensus sequences between Gonoloaeae strains are also shown.

  By way of further illustration, some representative NGDR9 related nucleic acid sequence variations are associated with gene ID numbers NG0465, NG0466, NG0467, IGR0389, IGR0390, NG1616, NG1617, NG1618, IGR1318, and IGR1319, and some representative NGDR33 Related nucleic acid sequence variations are associated with gene ID numbers NG0518, NG0519, NG0520, IGR0430, IGR0431, NG1649, NG1650, IGR1345, and IGR1346, all of which are dated March 12, 2004 to stdgen. lanl. In gov, it is provided on the World Wide Web.

Silent Variations Numerous nucleic acid sequences encoding NGDR9 and NGDR33 polypeptides may be produced by those skilled in the art due to the degeneracy of the genetic code, some of which are specifically disclosed herein. It will be appreciated that a minimal sequence homology may be sufficient. Representative NGDR9 polypeptides are associated with gene ID numbers NG0465, NG0466, NG0467, NG1616, NG1617, and NG1618, and representative NGDR33 polypeptides have gene ID numbers NG0518, NG0519, NG0520, NG1649, and NG1650. All of which are related to stdgen. lanl. In gov, it is provided on the World Wide Web.

  For example, examination of the codon table (Table 7) shows that the codons AGA, AGG, CGA, CGC, CGG, and CGU all encode the amino acid arginine. Thus, at every position of the nucleic acid of the invention where arginine is specified by a codon, the codon can be changed to any of the corresponding codons described above without altering the encoded polypeptide. is there. It is understood that U in the RNA sequence corresponds to T in the DNA sequence.

  Such “silent variations” are a type of “conservatively modified variations” disclosed below. One skilled in the art recognizes that each codon of a nucleic acid (other than AUG, which is usually the only codon for methionine) can be modified to encode a functionally identical polypeptide by standard techniques. Will do. Thus, each silent variation of a nucleic acid encoding a polypeptide is implied in any described sequence. The present invention provides each and all possible variations of nucleic acid sequences encoding NGDR9 and NGDR33 polypeptides, which are created by selective combinations based on possible codon choices. Is also possible. These combinations are made according to the standard triplet genetic code (eg, as shown in Table 1) applied to the nucleic acid sequences encoding NGDR9 and NGDR33 polypeptides. All such variations of each nucleic acid herein are clearly provided and described by consideration of the sequence in combination with the genetic code.

Conservative variation A “conservatively modified variation” or simply “conservative variation” of a particular nucleic acid sequence is a nucleic acid that encodes the same or essentially the same amino acid sequence, or a nucleic acid encodes an amino acid sequence. If not, it refers to an essentially identical sequence. One skilled in the art will alter, add, or add a single amino acid or a low percentage of amino acids (typically less than 5%, more typically less than 4%, 2%, or 1%) in the encoded sequence, or It will be appreciated that individual substitutions, deletions or additions that are deleted are “conservatively modified variations” (wherein the change results in an amino acid deletion, amino acid addition, Or substitution of amino acids with chemically similar amino acids).

  Conservative substitution tables providing functionally similar amino acids are known in the art. Table 8 shows six groups containing amino acids that are “conservative substitutions” for each other.

  Thus, a “conservative substitution variation” of NGDR9 and NGDR33 polypeptides referred to herein is a low percent, typically less than 5%, more typically less than 2% or 1% of the polypeptide. Substitution of amino acids of the sequence by amino acids of the same conservative substitution group selected conservatively.

  The addition of sequences that do not alter the activity of the nucleic acid molecule, such as the addition of non-functional sequences, is a conservative variation of each basic idea.

  One skilled in the art will recognize that many conservative variations of the nucleic acids described herein result in functionally identical nucleic acids. For example, as discussed in the preceding sentence, due to the degeneracy of the genetic code, a “silent substitution” (ie, a substitution in a nucleic acid that does not result in a change in the encoded polypeptide) An implicit feature of nucleic acid sequences. Similarly, a “conservative amino acid substitution” in which one or several amino acids in an amino acid sequence is replaced by another amino acid with highly similar properties is also highly similar to the disclosed construct. It is easily identified. Such conservative variations for each disclosed sequence are a feature of the present invention.

V. Probe and Primer Synthesis The oligonucleotide probes and primers of the present invention can be synthesized using essentially any technique known in the art. In some embodiments, for example, the oligonucleotide probes and primers described herein are described by Tetrahedron Letts., For example, by Beaucage and Caruthers, which are incorporated herein by reference. 1981, Vol. 22, No. 20, p. 1859-1862 chemically using the solid phase holamidite triester method or other synthetic techniques known in the art, such as Needham-Wande Wander, which is included herein as a reference. (Needham-VanDevanter) et al., Nucleic Acids Res., 1984, Vol. 12, p. It is synthesized using an automatic synthesizer as described in 6159-6168. A wide variety of equipment is commercially available for automated oligonucleotide synthesis. Multinucleotide synthesis methods (eg, trinucleotide synthesis, etc.) are also optionally utilized. Furthermore, the primer nucleic acids described herein optionally include various modifications. In certain embodiments, for example, a primer includes a restriction site linker, etc., for example, to facilitate subsequent amplicon cloning. To further illustrate, primers may also optionally improve the specificity of the amplification reaction, eg, “MODIFIED NUCLEIC ACID AMPLIFICATION PRIMERS, published December 14, 1999, which is incorporated herein by reference. Modified as described in US Pat. No. 6,001,611 to Will entitled “Modified Nucleic Acid Amplification Primer”. Primers and probes can also be synthesized using a variety of other modifications, as described herein, otherwise known in the art.

  Essentially any label is optionally utilized to label the nucleic acid detection reagent of the present invention. In some embodiments, for example, the label is a fluorescent dye (eg, a rhodamine dye (eg, R6G, R110, TAMRA, ROX, etc.), a fluorescein dye (eg, JOE, VIC, TET, HEX, FAM, etc.), a halogenated fluorescein dye, Cyanine dyes (eg CY3, CY3.5, CY5, CY5.5 etc.), BODIPY® dyes (eg FL, 530/550, TR, TMR etc.), ALEXA FLUOR® dyes (eg 488, 532, 546, 568, 594, 555, 653, 647, 660, 680, etc.), dichlororhodamine dye, energy transfer dye (eg, BIGDYE® v1 dye, BIGDYE® v2 dye, BIGDYE ( Registered trademark) v3 Etc.), Lucifer dyes (eg, Lucifer Yellow), CASCADE BLUE®, Oregon Green, and the like.Additional examples of fluorescent dyes include: For example, Haugland, “Molecular Probes Handbook of fluorescent Probes and Research Products”, 9th edition, 2003, and its latest edition, these Fluorescent dyes are typically included in, for example, Molecular Probes Inc. (Eugene, Oreg.), Amersham Biosciences Corp. (New Jersey) , Piscataway), Applied Biosystem (Applied Biosystems) (Foster City, Calif.), Etc. Other labels include, for example, biotin, weak fluorescent labels (Yin et al., Appl Environ Microbiol., 2003, 69, 7, p. 3938, Babendure et al., Anal. Biochem., 2003, 317, 1, p. 1, and Giancoviak (Jankowiak) et al., Chem Res Toxicol., 2003, Vol. 16, No. 3, p. 304), non-fluorescent labels, colorimetric labels, chemiluminescent labels, bioluminescent labels (Wilson et al., Analyst. 2003, 128, No. 5, p. 480, and Roda et al. , Luminescence, 2003, Vol. 18, No. 2, p. 72), Rahman label, electrochemical label, bioluminescent label (Kitayama et al., Photochem Photobiol., 2003, Vol. 77, Vol. 3, p.333, Arakawa et al., Anal. Biochem., 2003, Vol. 314, No. 2, p. 206, and Maeda, J. Pharm. Biomed. Anal. 2003, Vol. 30, No. 6, p. 1725), and alpha-methyl as described, for example, in US Provisional Patent Application No. 60 / 428,484, filed Nov. 22, 2002. -PEG labeling reagents included, these references Each has been included as a reference is. Nucleic acid labels are also described further below.

  Furthermore, essentially any nucleic acid (and virtually any labeled nucleic acid, whether standard or non-standard) can be obtained from The Midland Certified Reagent Company (The Midland Certified Reagent Company), The Great American Gene. Any of a variety of commercial sources such as The Great American Gene Company, ExpressGen Inc., Operon Technologies Inc., Proligo LLC, and many others To custom order (custom order) or standard order (standard order).

VI. Sample preparation and nucleic acid amplification. Gonoloae and / or C.I. Isolated from a subject suspected of having a Trachomatis infection, typically a mammalian subject, and more typically a human subject. Typical samples or specimens include blood, plasma, serum, urine, joint fluid, semen, seminal plasma, prostate fluid, vaginal fluid, cervical fluid, uterine fluid, cervical scrape, amniotic fluid, anal scrape, mucus, Includes coffins, tissues, and the like. Any technique can be used, including essentially any technique for obtaining these samples, such as scraping, venipuncture, wiping fluid, biopsy, or other techniques known in the art.

  Methods of classifying specimens, culturing cells, isolating and preparing nucleic acids from sources are known in the art, many of which are provided in the references and / or herein. Further described in the examples.

  To further illustrate, prior to analysis of the target nucleic acids described herein, these nucleic acids may be purified or isolated from a sample that typically contains a complex mixture of different components. . The cells in the collected sample are typically lysed to release the cell contents. For example, N.I. Gonorrhoeae and other cells are lysed by contacting them with various enzymes, chemicals, and / or lysed by other approaches known in the art, such as degrading bacterial cell walls Can be done. In some embodiments, the nucleic acid is analyzed directly in the cell lysate. In other embodiments, the nucleic acid is further purified or extracted from the cell lysate prior to detection. Essentially any nucleic acid extraction method can be used to purify the nucleic acid in the sample utilized in the methods of the invention. Representative techniques that can be used to purify nucleic acids include, for example, affinity chromatography, hybridization to probes immobilized on a solid support, liquid-liquid extraction (eg, phenol-chloroform extraction), precipitation ( E.g. use of ethanol), extraction with filter paper, extraction with micelle-forming reagents (e.g. cetyl-trimethyl-ammonium-bromide, etc.), to fixed intercalating dyes (e.g. ethidium bromide, acridine, etc.) Binding to silica gel or diatomaceous earth, adsorption to magnetic glass particles or organosilane particles under chaotropic conditions, and / or the like. Sample processing is also described, for example, in US Pat. Nos. 5,155,018, 6,383,393, and 5,234,809, each of which is included as a reference.

  To further illustrate, unmodified nucleic acids can bind to substances having a silica surface. A number of these processes, optionally adapted for use in carrying out the methods of the present invention, are described in the art. Illustrative examples include Vogelstein et al., Proc. Natl. Acad. Sci. USA, 1979, vol. 76, p. 615-619 describes the purification of nucleic acids from agarose gels in the presence of sodium iodide using ground flint glass. Marko et al., Anal. Biochem., 1982, Vol. 121, p. 382-387 describes the purification of nucleic acids from bacteria on glass dust in the presence of sodium perchlorate. In DE-A 3734442, which is included as a reference, nucleic acids are isolated on glass fiber filters. Nucleic acids bound to these glass fiber filters are washed and then eluted using methanol containing Tris / EDTA buffer. A similar approach is described in Jakobi et al., Anal. Biochem., 1988, 175, p. 196-201, which is included as a reference. In particular, Jacobi et al. Describe selective binding of nucleic acids to a glass surface in chaotropic salt solutions and separation of nucleic acids from impurities such as agarose, proteins, and cell debris. In order to separate the glass particles from the impurities, the particles can be centrifuged or the liquid can be drawn through the glass fiber filter. In addition, the use of magnetic particles to immobilize nucleic acids after precipitation by addition of salt and ethanol is described, for example, by Alderton et al., Anal. Biochem., 1992, 201, p. 166-169, and PCT / GB91 / 00212, both of which are included as references. Aggregates are separated from the original solvent by applying a magnetic field and performing one or more washing steps. After at least one washing step, the nucleic acid is typically dissolved in Tris buffer.

  For example, magnetic particles in a porous glass matrix coated with a layer containing streptavidin can also be utilized in certain embodiments of the invention. These particles can be used, for example, to isolate biotin-conjugated nucleic acids and proteins. Ferrimagnetic, ferromagnetic, and superparamagnetic particles are also optionally utilized. Magnetic glass particles and related methods that can be adapted for use in the practice of the methods described herein are also described, for example, in WO 01/37291, which is included as a reference.

  The most powerful and basic technique for collecting and detecting nucleic acids is nucleic acid amplification. In the present invention, the nucleic acid amplification of interest typically precedes or coincides with the detection of the DNA. In addition, the oligonucleotide probes described herein are also optionally amplified following, for example, chemical synthesis or the like. In some embodiments, the detectable signal is amplified using, for example, branched nucleic acids or other signal amplification formats known in the art.

  Amplification methods optionally utilized or adapted for use with the oligonucleotides and methods described herein include, for example, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand Includes various polymerase or ligase mediated amplification methods such as displacement amplification (SDA), nucleic acid sequence based amplification (NASBA), rolling circle amplification (RCA), and / or the like. Details on the use of these and other amplification methods can be found, for example, in “PCR Protocols A Guide to Methods and Application” by Berger, Sambrook, Ausubel, which are all included as references. "Introduction to PCR Protocols, Methods and Applications", edited by Innis, Academic Press, San Diego, CA, 1990 (Innis), Schweitzer et al., "Combining nucleic acid amplification and detection" And Detection Combination) ", Curr Opin Biotechnol., 2001, Vol. 12, No. 1, p. It can be found in various articles and / or various standard texts, including 21-27. Many available biology texts have also expanded the discussion on PCR and related amplification methods. Nucleic acid amplification is also described, for example, in U.S. Pat. No. 4,683,202 of Mullis et al. (1987) and by Sooknanan & Malek, Biotechnology, 1995, Vol. 13, p. 563, both of which are included as references. An improved method for amplifying large nucleic acids by PCR is described by Cheng et al., Nature, 1994, 369, p. 684, which is included as a reference. In some embodiments, double stranded PCR amplification is utilized to amplify the target nucleic acid. Double-stranded PCR amplification is described, for example, by Gabriel et al., “Identification of human remains by immobilized sequence-specific oligonucleotide probe analysis of mtDNA hypervariable regions I and II. Identification of human remains by sequence-specific oligonucleotide probe analysis), Croat. Med. J., 2003, 44, 3, p. 293, and La et al., “Development of a duplex PCR assay for detection of Brachyspira hyodysenteriae and Brachyspira pilosicoli in pig feces”. Development of Strand PCR Assay) ", J. Clin. Microbiol., 2003, 41, 7, p. 3372, both of which are included as references. Optionally, labeled primers (eg, biotinylated primers, Scorpion primers, etc.) are utilized to amplify nucleic acids in a sample, eg, to facilitate detection of amplicons and the like. The Scorpion primers are also described, for example, by Whitcombe et al., “Detection of PCR products using self-probing amplicons and fluorescence”, Nat Biotechnol., 1999, Vol. 17, No. 8, p. 804-807, which is included as a reference. Labeling is further described herein.

  Amplicons are recovered and purified from other reaction components by any of a number of methods known in the art, including electrophoresis, chromatography, precipitation, dialysis, filtration, and / or centrifugation. . Nucleic acid purification aspects are described, for example, by Douglas et al., “DNA Chromatography”, Wiley John & Sons, Inc., 2002, and Schott. ), “Affinity Chromatography: Template Chromatography of Nucleic Acids and Proteins”, Chromatographic Science Series No. 27, Marcel Dekker, 1984, all of which are included as references. In some embodiments, the amplicon is not purified prior to detection. Amplicon detection is described further below.

VII. Probe Analysis In certain embodiments of the invention, the oligonucleotide probes described herein are covalently or non-covalently attached to a solid support, which is then derived from a subject. Contact with a sample comprising the amplified and labeled nucleic acid. In other embodiments, the probes of the invention are provided free in solution. Essentially any substrate material is optionally adapted for use in these aspects of the invention. In some embodiments, for example, the substrate is fabricated from silicon, glass, or a polymeric material (eg, a glass or polymer microscope slide, a silicon wafer, etc.). Suitable glass or polymer substrates, including microscope slides, are commercially available from a variety of commercial manufacturers such as Fisher Scientific (Pittsburgh, PA) or the like. In some embodiments, the solid support in the present invention is a membrane. Suitable membrane materials include, for example, polyaramid membranes, polycarbonate membranes, porous plastic matrix membranes (eg, POREX® Porous Plastic), porous metal matrix membranes, polyethylene membranes, poly (vinylidene diful). Orido) membrane, polyamide membrane, nylon membrane, ceramic membrane, polyester membrane, polytetrafluoroethylene (TEFLON (registered trademark)) membrane, woven mesh membrane, microfiltration membrane, nanofiltration membrane, ultrafiltration membrane, Dialysis membrane, composite membrane, hydrophilic membrane, hydrophobic membrane, membrane based on polymer, membrane based on non-polymer, powdered activated carbon membrane, polypropylene membrane, glass fiber membrane, glass membrane, nitrocellulose membrane, cellulose Membrane, nitrocellulose membrane, cellulose acetate DOO film, polysulfone film, polyethersulfone film, optionally selected from a polyolefin film or other like. Many other membranous materials are P.I. J. et al. Widely from various commercial manufacturers such as Cobert Associates, Inc. (St. Louis, Missouri), Millipore Corporation (Bedford, Massachusetts), or the like It is commercially available. Other representative solid supports include, for example, ceramics, metals, resins, gels, plates, beads, microbeads (eg, magnetic microbeads), tubes (eg, microtubes), whiskers, fibers, combs, It is optionally used including single crystals and self-assembled monolayers.

  The oligonucleotide probes of the invention can be directly or indirectly (eg, via a linker such as bovine serum albumin (BSA) or the like), for example, any available chemical to the support. Or it is deposited by physical methods. A wide variety of linking chemistries are available for linking molecules to a wide variety of solid supports. More specifically, the nucleic acid is attached to the solid support by covalent bonds, such as by conjugation with a coupling agent, or by non-covalent bonds, such as electrostatic interactions, hydrogen bonds, or antibody-antigen coupling. , Or a combination thereof. Exemplary coupling agents include biotin / avidin, biotin / streptavidin, Staphylococcus aureus protein A / IgG antibody Fc fragment, and streptavidin / protein A chimera (included as references, Sano et al., Bio / Technology, 1991, Vol. 9, p. 1378), or derivatives or combinations of these agents. The nucleic acid may be attached to the solid support by a photocleavable bond, electrostatic bond, disulfide bond, peptide bond, diester bond, or a combination of these bonds. The nucleic acid is also optionally attached to the solid support by a selectively releasable bond, such as 4,4'-dimethoxytrityl or a derivative thereof. Derivatives that have been found useful are 3 or 4 [bis- (4-methoxyphenyl)]-methyl-benzoic acid, N-succinimidyl-3 or 4- [bis- (4-methoxyphenyl)]. -Methyl-benzoic acid, N-succinimidyl-3 or 4- [bis- (4-methoxyphenyl)]-hydroxymethyl-benzoic acid, N-succinimidyl-3 or 4- [bis- (4-methoxyphenyl)]- Including chloromethyl-benzoic acid and salts of these acids.

  As mentioned in the preamble, the oligonucleotide probe is optionally attached to the solid support via a linker between the nucleic acid and the solid support. Useful linkers include coupling agents for binding to other or additional coupling partners, as described above, or to allow attachment to a solid support to be cleavable.

  The cleavable attachment is between the probe and the solid support, for example, oligopeptides, oligonucleotides, oligopolyamides, oligoacrylamides, oligoethylene glycols, alkyl chains consisting of between about 6-20 carbon atoms, and Can be made by attaching cleavable chemical components, including combinations of: These components may be cleaved by, for example, chemicals, electromagnetic radiation, or enzymes. Typical enzymatically cleavable attachments include peptide bonds cleavable by proteases, phosphodiester bonds cleavable by nucleases.

  Chemicals such as β-mercaptoethanol, dithiothreitol (DTT), and other reducing agents cleave disulfide bonds. Other agents that may be useful include oxidizing agents, wettable powders, and other selectively active ingredients. Electromagnetic radiation, such as ultraviolet, infrared, and visible light, cleaves photocleavable bonds. The attachment may also be reversible, for example by using heat or enzymatic treatment or reversible chemical or magnetic attachment. Dissociation and attachment can be performed using, for example, a magnetic field or an electric field.

  Array-based hybridization can be used to detect the presence of many amplicons simultaneously, so Gonoloae and / or C.I. It is particularly suitable for the detection of Trachomatis nucleic acid. A number of array systems have been described, including those commercially available from commercial manufacturers such as Affymetrix, Inc. (Santa Clara, California, USA) and the like. It can be adapted for use according to the invention. Aspects of array construction and use are also described, for example, by Sapolsky et al., “High-throughput polymorphism screening and genotyping with high-density oligonucleotide arrays”. Genetic Analysis: Biomolecular Engineering, 1999, Vol. 14, p. 187-192; Lockhart, “Mutant yeast on drugs”, Nature Medicine, 1998, Vol. 4, p. 1235-1236; Fodor, “Genes, Chips and the Human Genome”, FASEB Journal, 1997, Vol. 11, p. A879; Fodor, “Massively Parallel Genomics ", Science, 1997, Vol. 277, p. 393-395; and Chee et al., “Accessing Genetic Information with High-Density DNA Arrays”, Science, 1996, 274, p. 610-614, all of which are included as references.

  Optionally utilized in addition to the probes and primers described above for carrying out the methods and other aspects of the present invention; Gonoloae and / or C.I. Other probes and primers for detecting Trachomatis nucleic acids are described, for example, in US Pat. No. 5,550,040 to Purohit et al. And US Pat. No. 6,090,557 to Weiss. Both of these are included as references.

VIII. Nucleic acid hybridization. Their targets of oligonucleotide probes Gonoloae and / or C.I. Hybridization to a Trachomatis nucleic acid is achieved by selecting suitable hybridization conditions. The stability of the probe: target nucleic acid hybrid is typically selected to be compatible with the assay and wash conditions, and the stable, detectable hybrid between the probe and target N.P. Gonoloae and / or C.I. It should be formed only with the trachomatis nucleic acid. Manipulation of one or more different assay parameters determines the exact sensitivity and specificity of a particular hybridization assay.

More specifically, hybridization between complementary bases of DNA, RNA, PNA, or a combination of DNA, RNA, and PNA includes temperature, salt concentration, electrostatic strength, buffer composition, and the like. It occurs under a wide variety of different and different conditions. Examples of these conditions and methods for applying them are typically described in, for example, the hybridizations described in Tijssen (1993), supra, and Hames Higgins, supra. Depending on the nature of the sequence to be hybridized and its length, it is carried out between about 0 ° C. and about 70 ° C. for about 1 minute to about 1 hour. However, hybridization can occur for seconds or hours, depending on the reaction conditions. Illustratively, typical hybridization conditions for a mixture of two 20-mers are to bring the mixture to 68 ° C, followed by room temperature (22 ° C) for 5 minutes, or 2 ° C in 2 microliters. Cool to a very low temperature. Hybridization between nucleic acids uses buffers such as Tris-EDTA (TE), Tris-HCl, and HEPES, salt solutions (eg, NaCl, KCl, CaCl ₂ ), or other aqueous solutions, reagents, and chemicals. May be promoted. Examples of these reagents include RecA protein, T4 gene 32 protein, E. coli. E. coli single chain binding protein, and main or minor nucleic acid groove binding protein. Other examples of such reagents and chemicals include divalent ions, multivalent ions and intercalating substances such as ethidium bromide, actinomycin D, psoralen, and angelicin. A representative hybridization method for use in the present invention is the COBAS AMPLICOR® Chlamydia trachomatis (CT) / Nicelia Gonoroae (NG) test protocol (Roche Diagnostics Corporation, Indiana). The conditions are similar to those specified in the State, Indianapolis).

IX. Detection and Probe Variation As mentioned in the preceding sentence, the amplified target N.D. in the sample utilized in the method of the invention. Gonoloae and / or C.I. The trachomatis nucleic acid is optionally labeled to allow detection of the oligonucleotide probe-target hybridization duplex. In general, a label can be any component that can be attached, for example, to a primer utilized for amplification and that provides a detectable signal (eg, a quantifiable signal). The label may be attached to the primer directly or jointly by a variety of techniques known in the art. Depending on the type of label used, the label can be attached to the terminal (5 'or 3' end of the primer) or non-terminal nucleotide, and linker or spacer arms of various sizes and compositions can be attached. It can be indirectly attached via. Using commercially available phosphoramidite reagents, oligomers containing functional groups (eg thiols or primary amines) at either the 5 ′ or 3 ′ end can be produced by appropriately protected phosphoramides. Such oligonucleotides are described, for example, in “PCR Protocols: A Guide to Methods and Applications” (edited by Innis et al., Academic Press, Inc., San Diego, Calif., 1990). It is possible to label using the protocol described in). In one embodiment, the label consists of biotin covalently attached to the primer at the 5 'end. The term “biotinylated primer” refers to a primer having one or more biotin molecules attached to the primer directly or indirectly through an intervening linker molecule.

  By way of further illustration, detection of oligonucleotide probe-target hybridization duplexes may optionally be performed, for example, by Whitehead et al., Nature, 1983, 30, No. 5, p. 158 and commercially available by a chemiluminescent assay using a luminol-based reagent. Following hybridization of the probe to the labeled target DNA, the biotin molecule attached to the target DNA is conjugated, for example, to streptavidin-horseradish peroxidase (SA-HRP). Alternatively, the target DNA can be directly labeled with horseradish peroxidase, thereby eliminating the separate conjugation step. In either case, subsequent oxidation of luminol by the horseradish peroxidase enzyme results in photon emission, which is then detected, for example, on standard autoradiographic film. The strength of the signal is a function of the amount of DNA. A series of DNA standards containing a known amount of DNA are typically assayed together with one or more unknown samples. The signal intensity of a known DNA standard allows an empirical measurement of the functional relationship between signal intensity and the amount of DNA, which allows the quantification of unknown samples. Many other detection methods are also optionally utilized to carry out the methods of the invention, are referenced in the references cited herein and / or are known in the art.

  N. Gonoloae and / or C.I. Any available method for detecting a Trachomatis amplicon can be used in the present invention. Common approaches include real-time amplification detection using molecular beacons or 5′-nuclease probes, detection of intercalating dyes, labeling incorporated into the amplification probe or amplified nucleic acid itself, e.g. Detection (following electrophoretic separation of the amplification product from the uptake label), hybridization-based assays (eg, array-based assays), and / or detection of a second reagent that binds to the nucleic acid. For example, NG and / or CT are detected in one aspect of the invention using the oligonucleotides described herein in an AMPLICOR® test format.

  To further illustrate, a molecular beacon or 5′-nuclease probe is optionally designed to contain an oligonucleotide of the invention (ie, selected from SEQ ID NOs: 3 to 27), and a molecular beacon or 5′-nuclease probe. N. Gonoloae and / or C.I. It can be used to detect a Trachomatis amplicon. Molecular beacons or 5'-nuclease probes are further described below. Details about these general approaches are found in the references cited herein, for example, Sambrook & Ausubel. Additional labeling strategies for labeling nucleic acids and corresponding detection strategies are described, for example, by Haugland, “Handbook of Fluorescent Probes and Research Chemicals”, 9th edition. , Molecular Probes, Inc. (Eugene, Oreg.), 2003, which is included as a reference.

  Molecular beacons (MB) are oligonucleotides designed for real-time detection and quantification of target nucleic acids (eg, target N. gonorrhoeae and / or C. trachomatis amplicons). The 5 'and 3' ends of the MB collectively comprise a pair of components that impart the detectable nature of the MB. One of the ends is attached to the phosphor and the other is attached to a quencher molecule capable of quenching the fluorescence emission of the phosphor. For example, one example of a phosphor-quencher pair could use a phosphor such as EDANS, eg, at the 5′-end, and a quencher such as Dabcyl, eg, at the 3′-end. is there. When MB is present free in solution, i.e. not hybridized to a second nucleic acid, the MB trunk is stabilized by complementary base pairing. This self-complementary pairing results in a “hairpin loop” structure in the MB, where the phosphor and the quenching component are proximal to each other. As a confirmation of this, the fluorescent component is quenched by the phosphor. Molecular beacon loops typically comprise an oligonucleotide described herein (ie, selected from SEQ ID NOs: 3-27 or complements thereof), and thus target N.D. Gonoloae and / or C.I. Complementary to the sequence to be detected in the Trachomatis nucleic acid, the hybridization of the loop to its complementary sequence in the target forces the stem to dissociate, thereby moving the fluorophore and quencher away from each other . This results in non-quenching of the phosphor and causes an increase in MB fluorescence.

  Details regarding standard methods of creating and using MBs are well established in the literature, and MBs are available from a number of commercial sources. Further details regarding the production and use of MB can be found in, for example, Leone et al., “Molecular beacon probes combined with amplification by NASBA enable homogenous real-time detection of RNA. Molecular Beacon Probe Combined with Nucleic Acids Res., 1995, Vol. 26, p. 2150-2155; Hshuih et al., “Novel, ligation-dependent PCR assay for detection of hepatitis C in serum,” J. Clin Microbiol, 1997, 34, p. 501-507; Kostrikis et al., “Molecular beacons: spectral genotyping of human alleles” Science, 1998, Vol. 279, p. 1228-1229; Sokol et al., "Real time detection of DNA: RNA hybridization in living cells" Proc. Natl. Acad. Sci. USA, 1998, Vol. 95, p. 11538-11543; Tyagi et al., “Multicolor molecular beacons for allele discrimination” Nature Biotechnology, 1998, Vol. 16, p. 49-53; Fang et al., “Designing a novel molecular beacon for surface-immobilized DNA hybridization studies”, J. Am. Chem. Soc. 1999, vol. 121, p. 2921-2922; and Marras et al., “Multiplex detection of single-nucleotide variation using molecular beacons” Genet. Anal. Biomol. Eng., 1999. Vol. 14, p. 151-156, all of which are included as references. An aspect of MB construction and use is also a US patent entitled “Detectably labeled dual conformation oligonucleotide probes, assays and kits” to Tyagi et al. No. 5,925,517 (July 20, 1997), Tyagi et al., “Nucleic acid detection probes having non-FRET fluorescence quenching and kits and assays including such probes (nucleic acid detection with non-FRET fluorescence quenching). US Pat. No. 6,150,097 (November 21, 2000) entitled “Probes and Kits and Assays Containing Such Probes”; “Wavelength-shifting probes and primers and their use in assays and kits” Wavelength-shifting probes and primers, and in assays and kits These applications) entitled "US Pat. No. 6,037,130 (also found in the patent literature, such as March 14, 2000), all of which are incorporated by reference.

  MB components (eg, oligos, including those labeled with fluorophores or quenchers) can be synthesized using conventional methods. Some of these methods are further described in the preamble. For example, oligonucleotides or peptide nucleic acids (PNA) can be synthesized using standard methods in a commercial automated oligonucleotide / PNA synthesizer. Labels can be attached to oligonucleotides or PNAs during automated synthesis or by post-synthesis reactions described above, for example, Tyagi & Kramer (1996). Year), see above. Aspects related to the synthesis of functionalized oligonucleotides are also described by Nelson et al., “Bifunctional Oligonucleotide Probes Synthesized Using A Novel CPG Support Are Able To Detect Single Base Pair Mutations. Bifunctional oligonucleotide probes are capable of detecting single base pair mutations) "Nucleic Acids Res., 1989, 17, p. 7187-7194, which is included as a reference. The label / quencher introduces, for example, a quencher (eg, 4-dimethylaminoazobenzene-4′-sulfonyl moiety (DABSYL)) to the oligonucleotide or PNA using, for example, a conditioned probe glass column. Can be introduced. For example, a quencher can be added to the 3 ′ end of an oligonucleotide during automated synthesis; the succinimidyl ester of 4- (4′-dimethylaminophenylazo) benzoic acid (DABCYL) is , Can be used when the addition site is a primary amino group; and 4-dimethylaminophenylazophenyl-4′-maleimide (DABMI) can be used when the addition site is a sulfhydryl group. is there. Similarly, fluorescein is introduced in an oligonucleotide, for example, using a fluorescein phosphoramidite that replaces the nucleotide with a fluorescein, or by using a fluorescein dT phosphoramidite that introduces a fluorescein moiety in the thymidine ring via a linker. It is possible. In order to link the fluorescein moiety to the terminal position, iodoacetamidofluorescein can be linked to a sulfhydryl group. Tetrachlorofluorescein (TET) can be introduced using 5'-tetrachloro-fluorescein phoramidite during automated synthesis. Other reactive phosphor derivatives and their respective attachment sites are succinimidyl esters of 5-carboxyrhodamine-6G (RHD) linked to amino groups; tetramethylrhodamine iodos linked to sulfhydryl groups An acetamide; an isothiocyanate of tetramethylrhodamine linked to an amino group; or a Texas red sulfonyl chloride linked to a sulfhydryl group. During the synthesis of these labeled components, if desired, the conjugated oligonucleotide or PNA can be purified by, for example, high pressure liquid chromatography or other methods.

  Cruachem (cruachem.com), Oswel Research Products Ltd. (UK; oswel.com), Research Genetics (Invitrogen Division, Huntsville, Alabama (resgen. Com)), The Midland Certified Reagent Company (the Midland) A variety of commercial manufacturers, including Certified Reagent Company (Midland, Texas, mcrc.com) and Gorilla Genomics LLC (Alameda, Calif.) Produce standard and custom molecular beacons. is doing. Sentinel® Molecular Beacon Allelic Discrimination Kits from Stratagene (La Jolla, Calif.) And Eurogentec SA (Belgium, eurogentec.com) Various kits utilizing molecular beacons are also commercially available, such as and various kits from Isogen Bioscience BV (Isogen.com, The Netherlands).

  In some embodiments, a real-time PCR assay system that includes one or more 5'-nuclease probes is amplified N.p. Gonoloae and / or C.I. Used for detection of trachomatis nucleic acid. These systems use the intrinsic nuclease activity of certain polymerases to cleave the quencher or label from the oligonucleotide of the invention comprising the quencher and label, resulting in non-quenching of the label. The polymerase cleaves the label only at the beginning of replication, ie, when the oligonucleotide is bound to the template and the polymerase extends the primer. Thus, an appropriately labeled oligonucleotide probe and a polymerase comprising the appropriate nuclease activity are of interest to N. cerevisiae. Gonoloae and / or C.I. It can be used to detect trachomatis nucleic acid. For example, real-time PCR product analysis by FRET or the like (and related real-time reverse transcription PCR) provides a well-known technique for real-time PCR monitoring that has been used in a variety of situations, which Can be adapted for use with the probes and methods described in the description (all included as references, by Laurendeau et al., “TaqMan PCR-based gene dosage assay for predictive testing in Individuals from a cancer family with INK4 locus haploinsufficiency (TaqMan PCR-based gene dosage assay for predictive testing in individuals from cancer families with INK4 locus haploinsufficiency) "Clin Chem, 1999, 45, 7 No., p. 982-6; Laurendeau et al., “Quantitation of MYC gene expression in sporadic breast tumors with a real-time reverse transcription-PCR assay. Clin Chem, 1999, Vol. 59, No. 12, p. 2759-65; and Kreuzer et al., “LightCycler technology for the quantitation of bcr / ab1 fusion transcripts” Cancer Research, 1999, 59th. Vol. 13, No. 13, p. 3171-4).

X. System The present invention also relates to N. Gonoloae and / or C.I. A system for detecting trachomatis is also provided. The system includes one or more nucleic acid detection reagents (eg, probe nucleic acids, sequence specific antibodies, etc.) described herein. In some embodiments, the nucleic acid detection reagents are arrayed on a solid support, while in others, they are provided in one or more containers, for example, for assays performed in solution. . The system also includes at least one detector (eg, a spectrometer, etc.) that detects binding between the nucleic acid from the sample and / or its amplicon and the nucleic acid detection reagent. Other detectors are further described below. In addition, the system also includes at least one controller operably connected to the detector. The controller includes one or more instruction sets that can detect the binding detected by the detector in the sample by Neisseria gonorrhoeae and / or C.I. Correlate with the presence of Trachomatis.

  In some embodiments, at least one container nucleic acid detection reagent. In these embodiments, the system further performs at least one temperature modulator connected to a vector container that regulates the temperature in the container, and / or, for example, one or more nucleic acid amplification techniques in the container. Optionally including at least one liquid transfer component (e.g., an automatic pipettor) for transferring liquid to and / or from the container.

  Using the nucleic acid detection reagent described in the present specification (for example, an oligonucleotide probe comprising a sequence selected from the group consisting of SEQ ID NOs: 3 to 27 or a complement thereof, a sequence-specific antibody, etc.) Gonoloae and / or C.I. A typical commercial system, optionally utilized to detect Trachomatis nucleic acid, is, for example, COBAS AMPLICOR (registered), commercially available from Roche Diagnostics Corporation (Indianapolis, Ind.). (Trademark) analyzer, LUMINEX 100 (R) system available from Luminex Corporation (Austin, TX), ABI available from Applied Biosystems (Foster City, CA) Includes PRISM® sequence detection system, and the like.

  The present invention further includes SEQ ID NO: 1, SEQ ID NO: 2, a variant thereof substantially identical that has at least 90% sequence identity with one of SEQ ID NO: 1 or 2, or Provided is a computer or computer-readable medium comprising a data set comprising a number of character strings corresponding to a number of sequences corresponding to a partial sequence of SEQ ID NO: 1, SEQ ID NO: 2, or the complement of said variant . Typically, at least one character string corresponds to a sequence selected from the group consisting of SEQ ID NOs: 3-27 or complements thereof. Typically, the computer or computer readable medium further includes an automatic synthesizer coupled to the output terminal of the computer or computer readable medium. The automatic synthesizer accepts instructions from a computer or computer readable medium, and the instructions direct the synthesis of one or more probe nucleic acids corresponding to, for example, one or more character strings in the data set. Exemplary systems and system components are further described below.

  The detector is configured to detect a detectable signal generated, for example, in another component of the system or proximal thereto (eg, in a container, on a solid support, etc.). In these systems, suitable signal detectors that are optionally utilized or adapted for use, for example, detect fluorescence, phosphorescence, radioactivity, absorbance, refractive index, luminescence, or the like. . The detector optionally monitors one or multiple signals, eg, upstream and / or downstream from the performance of a given assay step. For example, the detector optionally monitors multiple optical signals, which correspond to “real time” results in position. Exemplary detectors or sensors include photomultiplier tubes, CCD arrays, optical sensors, temperature sensors, pressure sensors, pH sensors, conductivity sensors, scanning sensors, or the like. Each of these as well as other types of sensors are optionally incorporated into the systems described herein. Optionally, the system of the present invention includes multiple detectors.

  More specific representative detectors that are optionally utilized in these systems include, for example, resonant light scattering detectors, emission spectrometers, fluorescence spectrometers, phosphorescence spectrometers, emission (cold light) spectrometers, spectrophotometers. Includes meters, photometers, and the like. For example, are various synthetic components for synthesizing the oligonucleotide probes described herein, including, for example, automated nucleic acid synthesizers, also available for use in the system of the present invention? Or adapted. Detectors and synthesis components optionally included in the system of the present invention are described, for example, by Skoog et al., "Principles of Instrumental Analysis", 5th edition, Harcourt Brace College Publishers. (Harcourt Brace College Publishers), 1998 and by Currell, "Analytical Instrumentation: Performance Characteristics and Quality", John Wiley & Sons, Inc. Inc.), 2000, both of which are included as references.

  The system of the present invention is also typically operatively connected to one or more components of the system (eg, detectors, composite components, thermal modulators, liquid transfer components, etc.) to control the operation of the components. Includes controller. More specifically, the controller typically receives, for example, data from the detector, affects the temperature in the container and / or adjusts the liquid flow to or from the selected container. Included as a separate or integral system component that is used to affect and / or regulate, or the like. The controller and / or other system component (s) may optionally be a suitably programmed processor, computer, digital device, or other information appliance (eg, analog to digital, or digital to analog, as appropriate) Instruct the operation of these devices, receive data and information from these devices, and interpret this information according to instructions programmed in advance or entered by the user (including converters) Functions to operate and report to the user. Suitable controllers are generally known in the art and are commercially available from a variety of commercial sources.

  An optional controller or computer optionally includes a monitor, which is often a cathode ray tube (“CRT”) display, a flat panel display (eg, active matrix liquid crystal display, liquid crystal display, etc.), or the like. Computer circuits are often placed in boxes, which include a number of integrated circuits such as microprocessors, memories, interface circuits, and others. The box also optionally includes a hard disk drive, a floppy disk drive, a high capacity removable drive such as a writable CD-ROM, and other general peripheral components. An input device such as a keyboard or mouse is optionally provided for input from the user. These components are further exemplified below.

  A computer is typically preprogrammed with user instructions, eg, in the form of user input into a set to parameter fields, eg in the GUI, or for a variety of different specific operations, for example. Appropriate software for receiving in the form of instructions. The software then translates these instructions into an appropriate language for instructing the operation of one or more controllers to perform the desired operation. The computer then receives data from, for example, sensors / detectors contained in the system and interprets the data. Follow the programming to supply it in a user-understood format or use that data to control the liquid flow regulator in response to liquid weight received from, for example, a weight scale or the like Start the controller instruction.

  The computer may be, for example, a PC (Intelx86 or Pentium (registered trademark) chip compatible DOS (registered trademark), OS2 (registered trademark), WINDOWS (registered trademark), WINDOWS NT (registered trademark), WINDOWS 95 (registered trademark), WINDOWS 98 (registered trademark). Trademark), WINDOWS 2000 (registered trademark), WINDOWS XP (registered trademark), LINUX-based equipment, MACINTOSH (registered trademark), Power PC, or UNIX (registered trademark) -based (for example, SUN (registered trademark) workstation) Device), or other commonly available computers known in the art. Word processing software (eg, Microsoft Word® or Corel WordPerfect®) and database software (eg, Microsoft Excel®, spreadsheet software such as Corel Quattro Pro®), or Standard desktop applications such as Microsoft Access® (spreadsheet software such as Paradox®) can be adapted to the present invention. For example, software for performing control of temperature modulators and liquid flow regulators can be used by those skilled in the art using standard programming languages such as Visual basic, Fortran, Basic, Java, or the like, Arbitrarily constructed.

  FIGS. 2 and 3 are illustrations illustrating an exemplary example system that includes a logic device in which various aspects of the invention may be embodied. From the teaching provided herein, the present invention is optionally implemented in hardware and / or software, as will be appreciated by practitioners of this technology. In some aspects, different aspects of the invention are implemented in either client-side logic or server-side logic. As will be appreciated in the art, the present invention or components thereof may be implemented in media program components (eg, fixed media components) that contain logic instructions and / or data, which are appropriately configured computer processing. When loaded into the device, the device is operated in accordance with the present invention. As will also be understood in the art, fixed media containing logic instructions may be delivered to the viewer on the fixed media for physical loading into the viewer's computer, or the logic instructions may be The contained fixed medium may reside on a remote server that is accessed via a communication medium for the purpose of downloading program components by an observer.

  In particular, FIG. 2 schematically illustrates a computer 200 with a detector 202 and a liquid transfer component 204 operatively connected thereto. Optionally, detector 202 and / or liquid transfer component 204 are operatively connected to computer 200 via a server (not shown in FIG. 2). During operation, the liquid transfer component 204 is typically labeled N.P. Gonoloae and / or C.I. A nucleic acid detection reagent array comprising a nucleotide probe, a sequence-specific antibody, etc. arrayed thereon, such as described herein, a liquid such as a sample aliquot comprising a Trachomatis amplicon Move to 206. Thereafter, the detector 202 typically adheres to the nucleic acid detection reagent array 206 after one or more washing steps have been performed to wash away non-hybridized nucleic acids from the nucleic acid detection reagent array 206 using the liquid transfer component 204. A detectable signal (eg, fluorescence emission, etc.) generated by a labeled amplicon that hybridizes to the labeled probe is detected. As additionally shown, the temperature modulator 208 is also operatively connected to the computer 200. Prior to performing the hybridization assay, the target N.P. Gonoloae and / or C.I. The Trachomatis nucleic acid can be amplified using a labeled primer nucleic acid (for example, a primer comprising a sequence selected from SEQ ID NOs: 3 to 27). The amplicons of these amplification reactions are then transferred to the nucleic acid detection reagent array 206 to perform a binding assay, typically using the liquid transfer component 204 as described above. In some embodiments, the binding assay can be performed using a molecular beacon, 5'-nuclease probe, or the like comprising a sequence selected from SEQ ID NOs: 3-27, such as N. Gonoloae and / or C.I. It is performed simultaneously with the amplification of the Trachomatis nucleic acid. In these embodiments, detector 202 detects the detectable signal produced when an amplification reaction is performed using temperature modulator 208.

  FIG. 3 schematically illustrates an information appliance or digital device 300 that can read instructions from media 302 and / or network port 304 that are optionally connectable to a server 306 having a fixed media 308. It may be understood as a possible logic device. The digital device 300 can then use these instructions to direct server or client logic to embody aspects of the present invention, as is understood in the art. One type of logic device that may embody the invention is a computer system such as that illustrated at 300, which includes a CPU 310, optional input devices 312 and 314, a disk drive 316, and an optional monitor 318. . Fixed media 302, or fixed media 308 on port 304, may be used to program such systems, such as disk-type optical or magnetic media, magnetic tape, solid dynamic memory or static memory, or the like. Represents a thing. In particular embodiments, the present invention may be embodied in whole or in part as software recorded on this fixed medium. Communication port 304 may also be used to initially receive instructions used to program such a system and may represent any type of communication connection. Optionally, the present invention may be embodied in whole or in part in an application specific integrated circuit (ACIS) or programmable logic device (PLD) circuit. In such cases, the present invention may be embodied in a computer understandable descriptor language, which may be used to create an ASIC, or PLD.

  FIG. 3 also includes an automatic synthesizer 320 that is optionally connected to the digital device 300 via a server 306. Optionally, the automatic synthesizer 320 is directly connected to the digital device 300. During operation, the automated synthesizer 320 typically provides instructions for synthesizing one or more primers or probes comprising a sequence selected from the group consisting of SEQ ID NOs: 3-27 or complements thereto. Receiving, it is placed in the data set by, for example, digital device 300 and / or computer readable media such as fixed media 302 and / or 308.

XI. Kit The nucleic acid detection reagent used in the method of the present invention is optionally packed in a kit. As described herein, the nucleic acid detection reagent of the present invention comprises SEQ ID NO: 1, SEQ ID NO: 2, a substantially identical variant thereof, and at least 90 of one of SEQ ID NO: 1 or 2. It binds detectably to a variant having% sequence identity, or a nucleic acid having a sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, or the complement of said variant. In addition, the kit also includes instructions for performing the appropriate packaging of reagents and materials and assays required for DNA immobilization, hybridization and / or detection, such as solid supports, buffers, enzymes, and DNA strands. May also be included. Optionally, the nucleic acid detection reagents (eg, oligonucleotide probes, sequence specific antibodies, etc.) of the present invention are provided already attached to a solid support or otherwise immobilized. As another option, the nucleic acid detection reagent is provided free in solution in a container, eg, for performing the detection method of the invention in solution phase. In some embodiments, the nucleic acid detection reagent of the kit includes a label and / or quencher, such as when a molecular beacon, 5′-nuclease, or the like comprises a sequence selected from SEQ ID NOs: 3-27. Char component. In certain embodiments, the kit further comprises target N.I. Gonoloae and / or C.I. It includes a labeled primer for amplifying the Trachomatis sequence.

  The kit also includes contacting one or more of: a nucleic acid detection reagent with a nucleic acid from a sample or an amplicon thereof, and a nucleic acid detection reagent; Gonoloae and / or C.I. Also included is a set of instructions for detecting binding between the Trachomatis nucleic acid, if any, for packaging the nucleic acid detection reagent and set of instructions, if any. Typical solid supports included in the kit of the present invention include, for example, plates, microwell plates, beads, microbeads, tubes (eg, microtubes), fibers, whiskers, combs, hybridization chips, membranes, single crystals. , Arbitrarily selected from ceramic layers, self-assembled monolayers, or the like.

  In some embodiments, the kit further includes, for example, N. N. for the amplification of a segment of gonoroae nucleic acid. It comprises at least one primer nucleic acid that is at least partially complementary to at least one segment of a Gonorrhoeae nucleic acid. In some embodiments, the kit also includes C.I. Also included is one or more primers for amplifying at least one segment of the Trachomatis nucleic acid. In these embodiments, the kit typically further comprises a set of instructions for amplifying one or more subsequences of a nucleic acid having one or more primer nucleic acids, at least one nucleotide incorporation biocatalyst, and 1 Or a plurality of nucleotides. In some embodiments, the primer nucleic acid comprises at least one label (eg, fluorescent dye, radioisotope). Suitable labels are further described herein. For example, the primer nucleic acid is optionally conjugated with biotin or a biotin derivative. In these embodiments, the kit typically further includes, for example, an avidin or avidin derivative, or a streptavidin or streptavidin derivative to achieve detection of binding between the nucleic acid detection reagent of the invention and the target nucleic acid. And the enzyme conjugated to. In these embodiments, the kit typically further comprises at least one nucleotide-loaded biocatalyst (eg, a polymerase, ligase, or the like). In these embodiments, the kit typically further comprises one or more nucleotides, eg, for use in amplifying the target nucleic acid. Optionally, at least one nucleotide comprises a label. In some of these embodiments, the kit can be further protected, for example, against at least one pyrophosphatase (eg, a thermostable pyrophosphatase), eg, carryover contamination, for use in minimizing pyrophosphorolysis. Uracil N-glycosylase (UNG) (eg, heat stable UNG) for use in applications.

XII. EXAMPLES It is understood that the examples described herein are for illustrative purposes only and are not intended to limit the scope of the claims. Also, various modifications or changes in light of the examples and embodiments described herein can be suggested to those skilled in the art, which are within the spirit and scope of this application, and the attached patents. It is also understood that it should be included within the scope of the claims.

Example 1 Neisseria gonorrhoeae direct repeat 9 Detection and synthesis of N. gonorrhoeae detection Neisseria gonorrhoeae direct repeat 9-specific oligonucleotide primers for PCR analysis Neisseria gonorrhoeae direct repeat 9 (NGDR9) is a gene in the genome of Neisseria gonorrhoeae. It was previously identified as a two-copy DNA sequence. The sequence of NGDR9 was obtained from the Los Alamos National Laboratory Sexually Transmitted Diseases database, stdgen. lanl. Obtained by the World Wide Web at gov / stdgen / bacteria / ngon /. The entire 806 base pair NGDR9 sequence lacks substantial identity with any sequence in the Neisseria gonorrhoeae genome, but Brucella suis 1330, chromosome I, compartment 155 (GenBank) (R) Accession No. AE014469) has a sequence identity of 36.85% with gap (44.4% identical without gap). FIG. 4 depicts a ClustalW alignment of the NGDR9 sequence using a portion of this Brucella sequence. The NGDR9 sequence is B.I. Complementation to upstream and downstream oligonucleotide primers (NG519 (5'-CTCTCAATGCCCCAATCATAAAGC-3 ') and NG514, scanned for regions of minimal sequence identity with Switzerland (B. suis), each spanning a 190 base pair region of NGDR9 (Ie, 5′-GATAAAAGCAGAGCGAAGCGGATAC-3 ′ (SEQ ID NO: 24)). The deoxycytidylate unit at the 3 ′ end of these primers is, for example, included as a reference, 1999 12 Contains t-butylbenzyl group as described in US Pat. No. 6,001,611 to Will entitled “MODIFIED NUCLEIC ACID AMPLIFICATION PRIMERS” issued 14 May NGD is modified accordingly. Position of the track the process by your NG519 and NG514 in 9 are underlined in FIG. 4.

Other representative upstream and downstream oligonucleotide primer pairs are also underlined in FIG. In particular, DK101 (5'-GTTTGGCGGCAGAGCATCT-3) and DK102 (5'-AAATGGGATGCTGTCGCAA-3 ') are shown. Primer pair DK101, and the complement to DK102 (ie, 5′-TTGACGACAGCATCCCATTT-3 ′ (SEQ ID NO: 25)) is designed to amplify the 416 base pair region of NGDR9. Primers corresponding to the complement to DK101 and DK102, further comprising a 5′-end restriction site linker, namely HINDDK101 (5′-GGC AAGCTT GTTTGGCGCAAGCATCT-3 ′; HindIII restriction site underlined), and BAMDK102 (5 '-GGC GGATCC TTGACGACAGCATCCCATTT-3'; the BamHI restriction site is underlined) was also synthesized. N. using this primer pair. A photograph of an agarose gel showing the detection of Gonoloaeae is provided below. DK103 (5′-AAACGCCAATTCTCAAACACCCTCA-3 ′) and DK104 (5′-TTTGACGGCCTCCACGCATAA-3 ′) are also underlined in FIG. The primer pair of DK103 and its complement to DK104 (ie, 5'-TTATGCGTGAGGCCGTCAAA-3 '(SEQ ID NO: 26)) is designed to amplify the 384 base pair region of NGDR9.

Neisseria Genomic DNA Purification Extraction of genomic DNA from various Neisseria strains was performed by using the PureGene® DNA purification system (Gentra Systems, Minneapolis, MN). Bacterial cells were grown on chocolate agar (Hardy Diagnostics, Santa Maria, Calif.) At 37 ° C. in 5% CO ₂ for 48 hours. Cells were separated from the agar surface, resuspended in PBS and centrifuged at 13,000-16,000 × g for 5 seconds to pellet the cells. The supernatant was removed by aspiration leaving behind 10-20 μl of residual liquid. The sample was vortexed vigorously and the pellet was resuspended in the residual liquid. DNA extraction was performed as directed by the manufacturer. Briefly, 300 μl of cell lysis solution was added to the resuspended cells and the cells were lysed by pipetting up and down. After addition of 1.5 μl RNase A solution to the cell lysate, the sample was mixed by inverting the tube 25 times and incubated at 37 ° C. for 5 minutes. The sample was cooled to room temperature by placing it on ice for 1 minute. To the lysate-treated cell lysate, a volume of 100 μl of Protein Precipitation Solution was added and the sample was mixed by vortexing vigorously for 20 seconds at high speed. The protein residue was precipitated by centrifugation at 13,000-16,000 × g for 1 minute. The supernatant containing the DNA sample was transferred into a clean 1.5 ml microcentrifuge tube, each containing 300 μl of 100% isopropanol. Samples were mixed by gently inverting the tube 50 times and then centrifuged at 13,000-16,000 × g for 1 minute. The supernatant was drained and the pellet was washed with 300 μl of 70% ethanol. The tube was again centrifuged at 13,000-16,000 × g for 1 minute. After removal of the supernatant, the tube was drained by inverting and the DNA pellet was resuspended in 50 μl Hydration Solution. Genomic DNA is quantified using PicoGreen double-stranded DNA quantitation reagents (dsDNA Quantitation Reagents (Molecular Probes, Eugene, OR)) and resuspended DNA is kept at -20 ° C until use. Stored.

Amplification of NGDR9 Segments Separate PCR reactions were performed using genomic DNA isolated as a template from various Neisseria species and a primer pair of primers, NG519, and complement to NG514 (above). PCR was performed using 50 mM Tricine (pH 8.3), 80 mM K (OAc) ₂ (pH 7.5), 2 mM Mn (OAc) ₂ (pH 6.5), 50 μM dATP, 50 μM dGTP, 50 μM dCTP, 100 μM dUTP, 20 U ZO5 DNA polymerase. (Roche Molecular Systems, Alameda, Calif.) Contains 5 U AmpErase® UNG (uracil-N-glycosylase), 0.5 μM of each primer, and 1 ng / μl ethidium bromide Performed in a volume of 100 μl. Genomic DNA was added as a template at 10 ³ genomic equivalents per reaction for Neisseria gonorrhoeae and 10 ⁶ genomic equivalents per reaction for Neisseria meningitidis and other Neisseria strains. The reaction was over 60 cycles consisting of denaturation at 95 ° C for 15 seconds, annealing at 58 ° C for 20 seconds, and final extension at 72 ° C for 5 minutes, COBAS TaqMan® PCR system (Roche Molecular Systems). (Roche Molecular Systems), Alameda, Calif.).

  A separate PCR reaction was also used to amplify the above 190 base pair segment of NGDR9 using genomic DNA isolated from various Neisseria species as a template and using primer pairs, HINDDK101 and BAMDK102 (above). This was done using a method similar to that used.

Analytical agarose gel electrophoresis of PCR products PCR products are added to 20 μl of DNA sample by adding 8 μl of 10 × gel loading buffer (0.025% bromophenol blue dye, 100 mM EDTA, and 30% sucrose). Prepared for gel electrophoresis analysis. Samples were then loaded with 3.0% (w / v) Nusieve, 0.5% (w / v) in 1X TB buffer (0.089 M Tris, 0.09 M boric acid, 2 mM EDTA, pH 8.0). An agarose gel was loaded into a horizontally submerged gel lane containing 0.5 μg / ml ethidium bromide. The electrophoresis running buffer was 1 × TB buffer containing 0.5 μg / ml ethidium bromide. The gel was run for 1 hour at 95-100 V and then removed and visualized on a long wave UV transilluminator. Specific gels were examined for the presence or absence of PCR products at the expected size of 190 or 416 bp.

  5A and B, and 6A and B are photographs of an agarose gel showing detection of a 190 base pair segment of NGDR9, whereas FIG. 7 is a photograph of an agarose gel showing detection of a 416 base pair segment of NGDR9. .

Example 2 Assay Illustrating Selective Detection of Gonorrhoeae This example includes primer nucleic acids NG519 (having a sequence corresponding to SEQ ID NO: 5) and NG514R (having a sequence corresponding to SEQ ID NO: 24), and a 5′-nuclease probe (sequence Having the sequence corresponding to number 18) alone or C.I. Includes use in combination with Trachomatis primate. In particular, the inclusivity of these assays (ie, the degree of ability to detect the target organism, N. gonorrhoeae in the sample) is illustrated in Table 9.

  The exclusivity of these assays (ie, the degree of ability to eliminate false positives when a species of Neisseria organism other than Gonorrhoeae is present in the sample) is illustrated in Table 10.

  The specificity of these assays (ie, the degree of ability to eliminate false positives when non-Neseria organisms are present in the sample) is illustrated in Table 11.

Example 3 Neisseria gonorrhoeae direct repeat 33 Selection and synthesis of Neisseria gonorrhoeae direct repeat 33-specific oligonucleotide primers for PCR primer analysis of Gonorrhoeae Neisseria gonorrhoeae direct repeat 33 (NGDR33) is a two-copy DNA sequence in the Neisseria gonorrhoeae genome. Identified earlier. The sequence of NGDR33 was obtained from the Los Alamos National Laboratory Sexually Transmitted Diseases database, stdgen. lanl. Obtained by the World Wide Web at gov / stdgen / bacteria / ngon /. A blast homology search for NGDR33 shows a number of significant hits with Neisseria meningitidis, and the entire 1142 base pair NGDR33 sequence is Meningitidis, serogroup B, strain MC58, compartment 77 (GenBank® accession number AE002435) has a sequence identity of 38.09% with gap (50.44% identical without gap) Have. FIG. A ClustalW alignment of the NGDR33 sequence is depicted using a portion of the Meningitidis sequence. The NGDR33 sequence is N.P. The upstream and downstream oligonucleotide primers (NG613 (5′-AATGTCGGGTTTGACGAAACTC-3 ′) and complements to NG614 (scanning for regions of minimal sequence identity with Meningitidis, each spanning the 265 base pair region of NGDR33) That is, 5′-AACGTCCGACAACCGGTAAC-3 ′) was synthesized.The positions of NG613 and NG614 are underlined in Figure 8. The deoxycytidylate unit at the 3 ′ end of these primers is described, for example, in the preamble As referenced, it has been modified to include a t-butylbenzyl group.

Purification, amplification and analytical agarose gel electrophoresis of Neisseria genomic DNA. Genomic DNA of various Neisseria species was purified as described in the preamble of Example 1. A separate PCR reaction was also performed using the genomic DNA isolated from various Neisseria species as a template and using the primer pair, NG613 and complement to NG614 (above), the 190 base pair segment of NGDR9 described above. This was done using a technique similar to that used to amplify. In addition, the PCR products of these amplification reactions were separated by electrophoresis as described in the preamble in Example 1. The gel was examined for the presence of a PCR amplification product at the expected size of 265 bp. FIGS. 9A and B and 10 are photographs of an agarose gel showing detection of this 265 base pair segment of NGDR33.

Example 4 N. in clinical samples. Gonoroae / C.I. Detection of Trachomatis This prophetic example is based on N. Gonoloae and C.I. Describes the protocol for detection of Trachomatis.

Clinical Samples Intracervical swab specimens from women and urethral swab specimens from men are collected by standard methods known in the art. Swabs can be prepared using suitable culture transport media (eg, 2SP, M-4 (Microtest, Inc., Atlanta, GA), Bartel's chlamydial (Intracel Corp.). Corp.), Isaqua, Washington, etc.), which is then used for PCR analysis (included by reference, Van der Pol et al., J. Clin. Microbiol). , 2000, vol. 38, pp. 1105-1112). These specimens are usually stored at 2-8 ° C. and are typically transported to the laboratory within 24-72 hours of collection. Specimens are typically vortexed with the swab in the tube, the cell culture is inoculated, and an aliquot of each specimen is transferred to a new tube, which is usually stored at 2-8 ° C. until 7 days after collection, then Processed for PCR analysis.

  Optionally, a 50 ml aliquot of the first urine is also collected from both men and women. Female urine specimens are collected before or after swab collection. Male urine specimens are collected after urethral swab specimens are obtained. Urine specimens are typically stored at room temperature and transported to the laboratory within 24 hours, or stored at 2-8 ° C., for example, if not transported within 24 hours of collection. Upon arrival at the laboratory, 500 μl aliquots are stored from the time of collection until 7 days, typically at 2-8 ° C., until processed for PCR analysis.

PCR analysis Each specimen is typically processed and subjected to either or both AMPLICOR® and COBAS AMPLICOR® tests as described in the manufacturer's package insert. For each sample processed, C.I. Trachomatis, N.M. The target DNA of Gonoloaeae and the internal control (IC) has at least two primer pairs, at least one C.I. A pair specific to Trachomatis and at least one N.I. Amplification simultaneously in a single reaction mixture containing a pair specific to Gonorrhoeae (eg comprising at least one sequence selected from SEQ ID NOs: 3 to 27). The resulting amplification products are usually captured separately and are Gonoloae (for example, comprising a sequence selected from SEQ ID NOs: 3 to 27), C.I. Microwell plates (AMPLICOR® format) coated with trachomatis and IC-specific oligonucleotide probes (included as references, by Crochfelt et al., “Detection of Neisseria gonorrhoeae and Chlamydia trachomatis in genitourinary specimens from men and women by a coamplification PCR assay ", J. Clin. Microbiol, 1997," Detection of Neisseria gonorrhoeae and Chlamydia trachomatis in urogenital specimens from men and women by co-amplification PCR assay " 35, p. 1536-1540) or by hybridization to magnetic microparticles (COBAS AMPLICOR® format). The COBAS AMPLICOR® analyzer automatically performs all of the amplification, hybridization, and detection steps (DiDomenico et al., “COBAS AMPLICOR®: a fully automated RNA and DNA amplification and detection system. for routine diagnostic PCR (COBAS AMPLICOR®: fully automated RNA and DNA amplification and detection for routine diagnostic PCR) ”Clin. Chem., 1996, Vol. 42, p. 1915-1923. Jungkind et al., “Evaluation of automated COBAS AMPLICOR PCR system for detection of several infectious agents and its impact on laboratory management”. Impact on room management) J. Clin Microbiol., 1 1996, 34, p. 2778-2783, both of which are hereby incorporated by reference. ”In the AMPLICOR® format, amplification is, for example, GeneAmp® PCR. The system 9700 thermal cycler (Perkin-Elmer (Norwalk, Conn.)) Is used and detection is performed manually (Loeffelholz et al., "Detection of Chlamydia trachomatis in endocervical specimens by polymerase chain reacion (Detection of Chlamydia trachomatis in cervical specimens by polymerase chain reaction) ") J. Clin, Microbiol., 1992, 30, p. 2847-2851, which is included as a reference).

Data analysis Specimens that produce a signal that exceeds a positive cut-off value (optical density [OD] of 2.0 (A ₆₆₀ ) for C. trachomatis and 3.5 (A ₆₆₀ ) for N. gonorrhoeae) are typically Is interpreted as positive for a particular organism, regardless of IC results. N. below a negative cut-off value (eg, OD of 0.2). Gonoloae or C.I. Specimens producing a Trachomatis signal are typically considered negative for a particular organism if the IC signal is above a specified cutoff (eg, OD of 0.2) and the test is considered valid. Interpreted. N. Gonoloae and C.I. Below the cut-off value and IC for both Trachomatis, Gonoloae or C.I. Specimens producing a Trachomatis signal are usually interpreted as inhibitory. Inhibitory specimens are typically retested by processing frozen aliquots of the original specimen. The results of repeated tests are classified using the above criteria.

  Specimens that produce results between negative and positive cutoffs (≧ 0.2, <3.5 for N. gonorrhoeae and ≧ 0.2, <2.0 for C. trachomatis), regardless of the IC signal, It is typically ambiguous about a particular organism. Ambiguous results are usually resolved by processing an aliquot of the original specimen, double retesting, and comparing the results to the initial test. These specimens show that if at least two reasonable tests show an N.D. of ≧ 2.0 for a particular organism. Gonoloae or C.I. If a trachomatis OD occurs, it is interpreted as positive for a particular organism. These specimens should be tested if at least two replicates show an N.D. of <0.2 OD for a particular organism. Gonoloae or C.I. If a trachomatis signal is generated, it is interpreted as negative for a particular organism if the IC signal exceeds the specified cutoff. If two replicates show an N.D. of <0.2 for a particular organism. Gonoloae or C.I. A specimen is usually interpreted as inhibitory if it produces Trachomatis OD and the IC signal falls below the specified cutoff for any of two replicates.

  Although the foregoing invention has been described in some detail for purposes of clarity and understanding, those skilled in the art will recognize, upon reading this disclosure, in form and detail, without departing from the true scope of the invention. It will be apparent that various changes can be made. For example, all the techniques and apparatus described above can be used in various combinations. All publications, patents, patent applications, and / or documents cited in this application are included as references for each purpose, each individual publication, patent, patent application, and / or document. To the same extent, all of which shall be incorporated by reference for all purposes.

Various N.P. It is explanatory drawing which showed the sequence alignment of Neisseria gonorrhoeae direct repeat 9 (NGDR9) using the arrangement | sequence of the amplicon of the genomic DNA from a Gonorrhoeae strain | stump | stock. Various N.P. FIG. 5 shows a sequence alignment of Neisseria gonorrhoeae direct repeat 9 (NGDR9) using the amplicon sequence of genomic DNA from the Gonorrhoeae strain. Various N.P. FIG. 5 shows a sequence alignment of Neisseria gonorrhoeae direct repeat 9 (NGDR9) using the amplicon sequence of genomic DNA from the Gonorrhoeae strain. Various N.P. FIG. 5 shows a sequence alignment of Neisseria gonorrhoeae direct repeat 9 (NGDR9) using the amplicon sequence of genomic DNA from the Gonorrhoeae strain. Various N.P. FIG. 5 shows a sequence alignment of Neisseria gonorrhoeae direct repeat 9 (NGDR9) using the amplicon sequence of genomic DNA from the Gonorrhoeae strain. Various N.P. FIG. 5 shows a sequence alignment of Neisseria gonorrhoeae direct repeat 9 (NGDR9) using the amplicon sequence of genomic DNA from the Gonorrhoeae strain. Various N.P. FIG. 5 shows a sequence alignment of Neisseria gonorrhoeae direct repeat 9 (NGDR9) using the amplicon sequence of genomic DNA from the Gonorrhoeae strain. Various N.P. FIG. 5 shows a sequence alignment of Neisseria gonorrhoeae direct repeat 9 (NGDR9) using the amplicon sequence of genomic DNA from the Gonorrhoeae strain. N. in the sample. 1 is a block diagram illustrating a representative example system for detecting Gonorrhoeae. FIG. FIG. 2 is a block diagram illustrating a representative example system that includes a computer and computer-related media in which various aspects of the invention may be embodied. ClustalW alignment of the NGDR9 sequence using part of the sequence of Brucella Swiss 1330, chromosome I, compartment 155 (GenBank® Accession No. AE014469). ClustalW alignment of the NGDR9 sequence using part of the sequence of Brucella Swiss 1330, chromosome I, compartment 155 (GenBank® Accession No. AE014469). ClustalW alignment of the NGDR9 sequence using part of the sequence of Brucella Swiss 1330, chromosome I, compartment 155 (GenBank® Accession No. AE014469). FIG. 5 is a photograph of an agarose gel showing detection of a 190 base pair segment of NGDR9. FIG. 5 is a photograph of an agarose gel showing detection of a 190 base pair segment of NGDR9. FIG. 5 is a photograph of an agarose gel showing detection of a 416 base pair segment of NGDR9. CristalW of Neisseria gonorrhoeae direct repeat 33 (NGDR33) using part of the sequence of Neisseria meningitidis, serogroup B, strain MC58, compartment 77 (GenBank® accession number AE002435) Indicates alignment. CristalW of Neisseria gonorrhoeae direct repeat 33 (NGDR33) using part of the sequence of Neisseria meningitidis, serogroup B, strain MC58, compartment 77 (GenBank® accession number AE002435) Indicates alignment. CristalW of Neisseria gonorrhoeae direct repeat 33 (NGDR33) using part of the sequence of Neisseria meningitidis, serogroup B, strain MC58, compartment 77 (GenBank® accession number AE002435) Indicates alignment. CristalW of Neisseria gonorrhoeae direct repeat 33 (NGDR33) using part of the sequence of Neisseria meningitidis, serogroup B, strain MC58, compartment 77 (GenBank® accession number AE002435) Indicates alignment. FIG. 3 is a photograph of an agarose gel showing detection of a 265 base pair segment of NGDR33. FIG. 3 is a photograph of an agarose gel showing detection of a 265 base pair segment of NGDR33.

Claims (4)

  An oligonucleotide comprising a nucleic acid sequence selected from SEQ ID NOs: 3 to 10, 13 to 25, 27 and their complements. A method for detecting Neisseria gonorrhoeae in a sample comprising:
(A) The nucleic acid derived from the sample is subjected to SEQ ID NOs: 3 to 10, 13 to 16, 24, 25, 27 and SEQ ID NOs: 3 to 10, 13 to 16, 24, 25, 27 in at least one nucleic acid amplification reaction. Contacting a first pair of primer nucleic acids consisting of a nucleic acid sequence selected from the group consisting of the complements of; and (b) said nucleic acid and / or one or more amplicons thereof during or after (a) Detecting Neisseria gonorrhoeae in the sample by detecting from the nucleic acid amplification reaction of
Comprising a method. A method for detecting Neisseria gonorrhoeae in a sample comprising:
(A) an oligo consisting of at least one oligonucleotide selected from the group consisting of SEQ ID NOs: 17 to 23 and complements of SEQ ID NOs: 17 to 23; Contacting with a nucleotide;
(B) monitoring the binding between the nucleic acid and / or its amplicon and the oligonucleotide,
Wherein the detectable binding between the nucleic acid and / or its amplicon and the oligonucleotide determines the presence of Neisseria gonorrhoeae in the sample. (A) at least one an oligonucleotide, SEQ ID NO: 3～10,13～25,27, and an oligonucleotide consisting of a sequence selected from the group consisting of the complement of SEQ ID NO: 3～10,13～25,27 And one or more of:
(B) To determine the presence of Neisseria gonorrhoeae in a sample that is unknown or unproven by monitoring the binding between the sample-derived nucleic acid and / or its amplicon and said oligonucleotide. Or (c) at least one container for packing at least one oligonucleotide;
A kit comprising:

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